Sage Journals: Discover world-class research

Abstract

Clinical laboratory medicine has seen the introduction and evolution of liquid chromatography tandem mass spectrometry in routine clinical laboratories over the last 10–15 years. There still exists a wide diversity of assays from very esoteric and highly specialist manual assays to more simplified kit-based assays. The technology is not static as manufacturers are continually making improvements. Mass spectrometry is now commonly used in several areas of diagnostics including therapeutic drug monitoring, toxicology, endocrinology, paediatrics and microbiology. Some of the most high throughput analyses or common analytes include vitamin D, immunosuppressant monitoring, androgen measurement and newborn screening. It also offers flexibility for the measurement of analytes in a variety of different matrices which would prove difficult with immunoassays. Unlike immunoassays or high-pressure liquid chromatography assays using ultraviolet or fluorescence detection, mass spectrometry offers better specificity and reduced interferences if attention is paid to potential isobaric compounds. Furthermore, multiplexing, which enables multiple analytes to be measured with the same volume of serum is advantageous, and the requirement for large sample volumes is decreasing as instrument sensitivity increases. There are many emerging applications in the literature. Using mass spectrometry to identify novel isoforms or modified peptides is possible as is quantification of proteins and peptides, with or without protein digests. Future developments by the manufacturers may also include mechanisms to improve the throughput of samples and strategies to decrease the level of skill required by the operators.

Keywords

Liquid chromatography tandem mass spectrometry applications future developments

Introduction

Mass spectrometry (MS) has grown in popularity in clinical laboratories over the last 10–15 years. The Association for Clinical Biochemistry and Laboratory Medicine (ACB) has recently supported members to set up a Specialist Interest Group for MS to reflect this increase in interest. A survey conducted in 2012 inviting labs to share their methodology and applications gave responses from 25 labs in the UK and Ireland reporting over 70 different applications (unpublished data).

The increase in popularity for quantitative assays is likely to be due largely to its perceived increase in specificity when compared to immunoassay and the scope for combining multiple analytes into one analytical run, also called ‘multiplexing’. However, this may raise some ethical considerations when measuring analytes that may not have been requested by the clinician. Another likely reason for the increase in interest in liquid chromatography tandem mass spectrometry (LC-MS/MS) assays over the last 10 years has been due to unexpected withdrawal of immunoassay equipment, kits or assays, leaving laboratories struggling to provide a service. An initial shift, particularly for vitamin D was seen in 2006 due to the withdrawal of the Nichols Advantage. More recently, customers of Siemens Healthcare Diagnostics have seen a withdrawal of an androstenedione assay. Often lot-to-lot variability of commercially available immunoassays can show significant shift, whereas in-house assays are more in the control of the user.

Despite these obvious advantages, its increased application has not been without problems. The high level of technical expertise required has been acknowledged by manufacturers resulting in modifications to software and the production of analytical kits.

Elements of LC-MS/MS operation

Ion production

Electrospray ionization (ESI)¹ and atmospheric pressure chemical ionization (APCI)² are the two most common ionization techniques used in clinical laboratories. In ESI, the eluent from the column passes through a stainless steel capillary to which a charge is applied. A heated gas, usually nitrogen, passes the outside of the capillary resulting in the solvent being nebulized at the end of the capillary (Figure 1). The mechanism for ion formation is not fully understood, and there are two proposed models. In the first model, the ion evaporation model,³ the droplet forms an ideal state to assist in desorption of solvated ions. The second is the charged residue model.⁴ In this, the charge is carried by the solvent and as the solvent evaporates, the charge remains on the gaseous analytes. The conditions and solvents can be optimized for the formation of positive or negative ions. APCI utilizes a plasma region for desolvation and is considered to be a harsher ionization technique; however, it is felt that this is more efficient at removing matrix components.

Figure 1.

Ion formation. The analytes of interest are present in the mobile phase and are nebulized using heated nitrogen gas to form a spray at the capillary tip to give charged droplets. The charged droplets undergo coulomb fission and result in smaller droplets. The final ion formation mechanism is unclear; however, the two main theories are depicted: the ion evaporation model and the charged residue model.

Fragmentation

After the parent ion has been selected by the first quadrupole, the ions enter a collision cell for fragmentation (Figure 2). The collision cell design varies between manufacturers, but all include the presence of a collision gas, usually argon or nitrogen. Kinetic energy is applied to the collision gas, and collision of the analyte with the gas molecules results in fragmentation.

Figure 2.

Selected reaction monitoring. At the first quadrupole (Q1), only the analyte of interest has the correct mass-to-charge ratio and hence oscillation to progress to the collision cell. In the collision cell, the analyte collides with a gas such as argon or nitrogen to induce fragmentation. The subsequent fragments are then subjected to selection by a second quadrupole with the selected mass to charge fragment reaching the detector.

Transitions and scans

Quadrupoles operate on the basis of mass-to-charge ratio, referred to as m/z. After fragmentation, the second quadrupole will also isolate a product ion based on its m/z. This is termed the ‘transition’, e.g. cortisol has a mass of 362.4 and gains a proton to form a positive ion with a mass of 363.4. It fragments in a collision cell to give multiple product ions, one of which has a mass of 97.0. As both carry one charge, the transition is referred to as m/z 363.4 > 97.0. Monitoring a single transition is called ‘selected reaction monitoring’ (SRM).⁵

Mass spectrometers have the ability to monitor multiple transitions rapidly in sequence. These may include an analyte and its internal standard or also include other analytes and their internal standards. This is called ‘multiple reaction monitoring’ (MRM) and forms the basis of most quantitative assays.

LC-MS/MS may also be used in scan mode.⁵ Most manufacturers can combine scans with quantitative LC-MS/MS for confirmation. Some manufacturers now offer equipment specifically designed to combine quantitation and confirmation with the introduction of an ion trap. Scans that can be performed on most LC-MS/MS include precursor scans, product ion scans or neutral loss scans.⁵

Sample preparation

A wide variety of sample preparation techniques are used for the pretreatment of samples prior to LC-MS/MS analysis. A simple protein precipitation is used for many assays such as immunosuppressants.

This typically consists of the lysis of whole blood with zinc sulphate^6,7 or by the addition of water followed by zinc sulphate.⁸ It is thought that the addition of water prevents clumping of red blood cells, thus causing an improved lysis and a cleaner supernatant.⁸ Solvent precipitation then follows by the addition of acetonitrile^6,7 or methanol.^9–11 This level of sample preparation is not dissimilar to the preparation required for semi-automated immunoassay methods for immunosuppressants that were used in the majority of laboratories before the widespread adoption of LC-MS/MS.

These techniques or similar are also adaptable to serum and plasma analytes and provides quick and simple sample preparation when sample dilution is acceptable. As more sensitivity or cleaner extracts are required, other sample preparation techniques may be utilized that allow for sample extraction and/or concentration, e.g. solid-phase extraction (SPE),^12,13 supported liquid extraction¹⁴ (SLE) and liquid-liquid extraction (LLE).^15–17

Derivitization is commonly associated with gas chromatography techniques but is still applicable for increasing sensitivity in LC-MS/MS analysis. Although derivitization is used rarely among routine clinical LC-MS/MS assay, this and other more involved sample preparation still has a role for some analytes that are more difficult to measure.^18,19 A recently published review demonstrated the importance and diversity of sample preparation.²⁰

Kits and calibration

A limitation of quantitative MS is the restricted availability of commercial calibrators, and these still need to be prepared in-house for many assays. There are several suppliers of immunosuppressant and vitamin D calibrators. In the case of vitamin D, harmonization of the calibrators used has shown to decrease significantly the assay-to-assay variation seen between LC-MS/MS users,^21,22 highlighting the difficulties of preparing them in-house. One source of variation for endogenous compounds may be the chosen matrix as matrix matching proves difficult. Several approaches have been used, e.g. methanolic,²³ phosphate-buffered saline based,^17,24 human serum or plasma,^25,26 charcoal stripped serum²⁷ or animal serum.²⁸ One study has shown that if sufficient attention is paid to the removal of matrix effects and ion suppression caused by matrix components, that matrix matching of calibrators is not necessary.²⁹

Manufacturers have begun to offer kits for the analysis of panels of tests such as immunosuppressants,^30,31 steroids³² and vitamin D metabolites.³³ These offer a complete ‘out of the box’ solution that are usually supported by the manufacturer, thus reducing the expertise required for developing and maintaining an in-house assay. The disadvantages may include wide lot-to-lot variation as seen in immunoassay, and these kits would typically be considerably more expensive than an in-house assay.

Column choice and ion suppression

The choice of chromatography column is arguably the most critical part of LC-MS/MS method development. The selected analytical column needs to have the appropriate selectivity to separate the analyte of interest from isobaric interferences or from any non-specific interferences that can be visualized on the baseline.³⁴ As well as these requirements, the column must also separate the analyte of interest away from any compounds that may cause suppression of ionization not visible as interferences on the baseline. Specific experiments must be conducted to identify where these compounds elute^35,36 (Figure 3). Compounds that may cause ion suppression include matrix components such as salts and phospholipids^35,37,38 or compounds that may be introduced into the assay such as slipping agents, e.g. oleamide from the plastics used in blood and serum collection and storage.³⁸ Internal standards are used during the sample preparation and may compensate for some matrix effects seen. The choice of internal standard is important as even if deuterated stable isotope is used matrix effects, referred to as differential matrix effects may be observed.³⁹ Sample clean-up techniques such as protein precipitation, SPE, LLE, SLE, phospholipid removal plates or turbulent flow are often used to try to remove compounds that cause ion suppression.⁴⁰ Despite this, their removal is usually not complete by these processes, and chromatography plays a major role. Combining the separation required without creating excessive run-times is a challenge.

Figure 3.

An example of an ion suppression infusion experiment chromatogram for prednisolone analysis. In (b), a constant infusion of prednisolone is entering the mass spectrometer as an extracted sample is being injected. An illustrative prednisolone peak is given in (a) for comparison. Ion suppression can be observed as reduction in the baseline in (b), as is seen at 0.5 min, highlighting that other compounds are elution that are competing for the limited ion formation capacity.

There is a large variety of chromatography columns available from each manufacturer, and this range is constantly evolving. In our experience, there are often differences between manufacturer’s products even with the same packing material. For example, two manufacturer’s silica-based columns with C18 coverage can show vastly different retention capacities and selectivity; one contributing factor is carbon loading. There have also been new developments in column technology in recent years, such as the emergence of fused core column technologies which propose faster re-equilibration times and therefore reduced run-times.⁴¹

Standardization and reference method services

There is considerable heterogeneity between LC-MS/MS laboratories when measuring the same analyte. For 25-hydroxyvitamin D analysis, there can be different equipment, calibrators, internal standards, sample preparation, chromatography and transitions. Any of these factors can contribute to interlaboratory variation with differing extents.⁴² For example, the use of common calibrators has been shown to significantly reduce the interlaboratory variation between LC-MS/MS users, suggesting that it was the preparation of calibrators that had the greatest effect.²¹ However, for testosterone, the use of a common calibrator did not show significant reduction in interlaboratory coefficients of variation (CVs), and it was proposed that it was methodology differences that were responsible.⁴³

Standardization efforts are currently limited in the area of quantitative LC-MS/MS due to the wide variety of assays and matrices utilized. Even when a reference method exists, it is difficult for National Health Service laboratories to take part in method comparisons with reference laboratories or utilize matrixed reference panels due to expense. An ideal and affordable way for labs to compare their assays to reference methods is the use of external quality assurance (EQA) material with assigned reference values such as the NIST assigned values to vitamin D EQA since 2013.⁴⁴ The Wales External Quality Assurance Scheme also offers reference method services. The onus is now on EQA providers to make this a more widespread activity.

Isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) is a technique that has recently grown in popularity for those developing reference methods but has been discussed in the reference method literature for at least 20 years⁴⁵ and was first proposed as an analytical tool in 1954.⁴⁶ As well as reference methods being available for analytes that are routinely measured by LC-MS/MS such as steroid hormones^17,27,47,48 and amino acids,^49–51 there are also methods for analytes for use as a reference method alone. These analytes include glycosylated haemoglobin (HbA1c),^52,53 cholesterol^54,55 and free thyroid hormones^56–62 among others. It should be noted that the isotope dilution technique is quite distinct from the routine use of stable isotope internal standards, although the term is often used interchangeably.

Internal standard choice

One of the areas that has changed since the methods were first reported is the area of internal standards. Original assays developed often used analogues, e.g. cyclosporin D for cyclosporin A^7,8,13,63,64 analysis and ascomycin for tacrolimus.^6,8,10 Typically, analogues are not as good as stable isotopes although ascomycin is still regarded as a good internal standard in tacrolimus measurement. These were later superseded by the availability of stable isotope internal standards.^31,65,66 Internal standards should be added to the sample early in the sample preparation protocol. They are present to account for any loss of sample during the extraction process and for any variation in ionization at the mass spectrometer source. To do this effectively, they must elute at the same time as the analyte of interest which was not always possible for analogues. Isotopes are more likely to behave similarly to the compound of interest during the extraction and ionization processed than analogues and should therefore best represent any losses.^67,68 They will also fragment in the collision cell in the same manner as the compound of interest.

The stable isotope most commonly used is deuterium as this is easy to incorporate and inexpensive. However, loss of label can occur due to deuterium exchange with hydrogen.^69,70 It is also possible to chromatograph a heavily labelled deuterium internal standard away from the compound of interest.³⁷ Carbon 13 isotopes give highly pure internal standards that are chemically stable with the same elution as the compound of interest,³⁷ e.g. ¹³C3 testosterone-2, 3, 4. Nitrogen 15 internal standards have similar advantages as carbon 13; however, both require a more complicated synthesis and are usually more expensive. An example of the use of a stable nitrogen isotope would be ¹³C-¹⁵N-Angiotensin I in the LC-MS/MS measurement of renin activity.^71,72

There are stable isotope internal standards available for most common applications, and the user may even have a choice, e.g. D2 testosterone-1,2 or D3 testosterone-16, 16, 17 or D5 testosterone-2, 2, 4, 6, 6 or ¹³C3 testosterone-2, 3, 4. If no carbon 13 isotope is available and there is a selection of deuterium isotopes available, the location of the deuterium atoms on the molecule will determine how likely they are to exchange, so users should discuss this with the manufacturers prior to selection. Generally speaking, carbon 13 or nitrogen 15 isotopes should be the internal standard of choice compared to D2 isotopes when setting up a new assay. Care should also be taken if considering a D2 isotope of a carbon-rich molecule such as testosterone due to the effects of natural isotopes.⁷³

Without the use of a suitable internal standard, measurements may be prone to unpredictable ion suppression matrix effects from samples or plasticizers introduced during sample preparation stages.³⁵

Matrices

MS gives the user the flexibility to measure analytes in alternative matrices for which it would prove difficult to use a kit method which may not be fully validated. Drug analysis is commonly carried out on hair samples as drugs can be detected in hair for much longer time periods than in other matrices and hair analysis can also give information on previous drug abuse.^74–77 Saliva sampling is viewed as less invasive than blood sampling and can be very useful for getting multiple samples over the course of time. Examples include assessing the adequacy of hydrocortisone^78,79 or testosterone¹⁶ replacement, or for obtaining late night or early morning samples for analytes that show circadian variation.^80,81 Dried blood spot analysis has been used for many years for newborn screening^82–84 and is now used for many different LC-MS/MS assays due to the increased stability of many analytes in dried blood spots compared to whole blood or serum.⁸⁵ Other matrices for which MS assays are reported include fibroblasts,⁸⁶ bile,⁸⁷ tears⁸⁸ and cerebral spinal fluid.^89,90

Clinical MS is constantly evolving and is applicable to lots of different areas of laboratory medicine.

Current clinical uses of MS

Therapeutic drug monitoring

One of the first uses of quantitative LC-MS/MS was for the therapeutic drug monitoring (TDM) of immunosuppressants. Cyclosporin and tacrolimus were the first assays to be developed, and many of the methods in use have changed little since they were first reported.

There are still at least seven immunoassays in use as reflected in EQA schemes. Some show distinct differences to MS and bias when compared to the target concentrations. This may be due to differences in specificity and/or calibration. Some immunoassays also demonstrate high between-laboratory CVs, up to around 15%, whereas MS is approximately half of this at around 8% even though different instruments, chromatography and calibration techniques will be in use for cyclosporin (Analytical Services International Proficiency Testing Scheme, Distribution 360). At least four other immunoassays are available for tacrolimus and some demonstrate poorer interlaboratory CVs at almost 26%; however, numbers are fewer. MS users on a recent distribution have a CV of 12.1% for a sample at the lower end of the therapeutic range (Analytical Services International Proficiency testing scheme distribution 226). Recent studies have shown that this interlaboratory CV can be improved further for MS if common platforms and calibrators are used.⁹¹

One major advantage of LC-MS/MS over immunoassay in the measurement of immunosuppressants is the ability to multiplex analysis of multiple analytes in one analytical run. Cyclosporin and tacrolimus are commonly combined into one assay,⁶³ and sirolimus and everolimus and/or mycophenolic acid may also be included.^10,92–94 MS multiplexing also gives the ability to measure related drugs such as everolimus for which there is no immunoassay readily available for the major immunoassay platforms.

LC-MS/MS offers users the flexibility to select different sampling strategies due to its low sample volume requirement. Fingerprick sampling has been used reliably for many years^7,95–98 and makes a significant difference to patient care as they can collect the samples themselves at home and post to the laboratory. This prevents regular journeys to transplant centres for long-term follow-up. Later studies have demonstrated the use of dried blood spot collection for long-term sample stability and avoidance of clotting.^99–104 One further reason for the popularity of immunosuppressant assays among LC-MS/MS users is the availability of commercially produced calibrators. Early assays involved calibrator production in-house using whole blood with added drug to give target concentrations^64,105 which has obvious ethical and health and safety implications. Commercial calibrator sets became readily available quickly negating the need for this process, and more recently complete kits have become available.^30–33

Immunosuppressants are not the only drug group for which LC-MS/MS is used for TDM; however, they are the largest group. Other uses include the measurement of antiepileptic drugs,^106–110 antibiotics^79,111,112 and antifungal drugs,^113–116 all of which may be as single assays or as part of a larger multiplexed panel. The details of these assays have been covered by a recent review.¹¹⁷ There are some assays also reported for more esoteric analytes such as ribavirin^82,118–120, clozapine,^121–125 imatinib,^126–131 busulfan,^132,133 caffeine^134,135 and amiodarone.^136,137

Toxicology

For many years, gas chromatography mass spectrometry (GC-MS) was the technique of choice for toxicological analysis due to its ability to screen for unknown drugs as well as to perform targeted screening for specific drugs. Immunoassay is commonly used in many laboratories as a first-line screening test, but any positive results must be confirmed by a second method, such as gas chromatography (GC) or LC-MS/MS. Immunoassays are also notorious for producing false-negative as well as false-positive results.¹³⁸ Although GC-MS is still commonly in use in many toxicology laboratories, the complexity of sample preparation, which usually involves time-consuming derivitization, has prompted many laboratories to move over to LC-MS/MS analysis.⁷⁶

LC-MS/MS can be used to screen for the presence of specific drugs in matrices such as urine, hair or saliva, by using MRM or SRM mode. Such an approach can be used to screen for multiple targeted analytes in one mass spectrometric assay.¹³⁹ A limitation of this approach is that it is unable to detect other substances that are present which are not part of the panel. Quantification of drugs can also be carried out using this method.¹⁴⁰ For a more systematic approach to identify the presence of drugs in a sample, many laboratories carry out a survey scan using MRM, followed by an enhanced product ion scan as a dependent scan.^141–144 Drug identification is then carried out by spectral comparison with a product ion spectral library, and such methodology can identify hundreds of drugs in a single LC-MS/MS run.¹⁴¹ This has become increasingly important in toxicology laboratories due to the emergence of unregulated psychoactive drugs, also known as legal highs.¹⁴⁵ High-resolution MS has been shown to be a valuable tool for this approach, the accurate mass and corresponding empirical formula of the unknown compound can be determined. This gives greater confidence in drug identification compared to lower resolution instruments.¹⁴⁶ A drawback of this approach is that isomers such as morphine and hydromorphone cannot be distinguished from one another. This problem may be overcome by producing collision-induced dissociation (CID) spectra of the compound of interest,¹⁴⁷ in combination with accurate mass and relative retention time to confirm the identity of the compound. An alternative approach, ion mobility, may be used to further distinguish these difficult analytes (Figure 4). Ion mobility is the separation of ions in the gas phase and is often coupled to time of flight instrument to provide separation of analytes prior to MS detection.

Figure 4.

Ion mobility. Ions enter a drift tube where they are exposed to an electric field. The drift tube also contains a drift gas that travels in the direction opposite to the ions. These conditions cause the ions to separate according to size as the larger the ion, the more the drift gas impedes its progress through the drift tube.

Endocrinology

LC-MS/MS is currently playing a major role in the area of endocrinology, particularly steroid hormone measurement and is useful as panels or related steroids may be measured simultaneously. Assays exist for testosterone,^{17,27,47,148,149} androstenedione,^17,47,48,149 dehydroepiandrosterone (DHEAS),^149,150 vitamin D,^151–154 oestrogens,^155–159 17 hydroxyprogesterone,^48,149,160 serum^24,161,162 and urinary cortisol^{161,163–166} among many more. The structural similarity between steroid hormones has been known for a long time to cause non-specificity issues for immunoassays, a well-documented example being DHEAS interference in testosterone immunoassays.¹⁶⁷ This can be observed as marked differences in method comparisons between immunoassay and LC-MS/MS or when reviewing EQA data, as method means for some immunoassays are 40% or greater than MS. There may be a case for measuring male testosterone by well validated immunoassays, but LC-MS/MS has also demonstrated superior performance at low concentrations for many analytes, e.g. female testosterone and oestradiol.^159,168 LC-MS/MS may also give more clinical information over immunoassay in certain situations such as serum cortisol measurement in patients taking metyrapone¹⁶² or prednisolone or the simultaneous measurement of dexamethasone and cortisol during suppression testing.¹⁶⁹ The use of LC-MS/MS in steroid hormone measurement has achieved recognition by clinical peer-reviewed journals such as the Journal of Clinical Endocrinology and Metabolism who published a position statement in 2013¹⁷⁰ requiring that any manuscripts presented to their journal from 2015 onwards will only be considered for publication if any sex steroid hormone determination is performed by MS-based methods. However, this is a controversial subject that is open to debate, and this decision has been suspended while an additional scientific review is conducted.¹⁷¹

Despite its generally accepted advantages, the measurement of steroid hormones by LC-MS/MS is not without problems. Some steroids have the same mass and fragment to give the same product ions and could therefore cause an isobaric interference. An example of this is 11-deoxycortisol, 21-deoxycortisol and corticosterone.¹⁷² In these cases, careful attention to chromatography should be applied to ensure adequate resolution of these compounds. The carbon-rich nature of steroids gives rise to another type of isobaric interference due to the so called ‘mass (M)+2’ effect.¹⁵⁹ This refers to natural isotopes of steroids containing two carbon 13 atoms having the potential to interfere in the measurement of a compound which has a mass 2 Daltons higher. An example of this would be oestradiol and oestrone. Oestradiol has a mass of 272.4 Daltons and oestrone 270.4 Daltons, both fragment to give the same product ion. A natural isotope of oestrone containing two carbon 13 atoms would have the same mass as oestradiol. Although this interference would be small, as oestrone concentrations in serum are greater, it should still be avoided by adequate chromatographic separation of the two.

Specific binding proteins for steroids have the potential to cause variability in their measurement. For example, immunoassays may have differing abilities to remove cortisol from cortisol-binding globulin, as demonstrated by under recovery in female subjects taking the oral contraceptive pill (OCP) compared to non-OCP users.¹⁷³ On the other hand, as MS methods usually involved protein precipitation and/or extraction, they can therefore avoid significant binding protein-related differences.²⁴ Solvents used in the extraction can be tailored for optimal extraction efficiency.

One difficulty in the use of LC-MS/MS in the measurement of steroids arises when their concentrations are in very low picomolar concentrations, such as oestradiol and aldosterone. Radioimmunoassays have the ability to amplify signal to enable the measurement of these two steroids.^174–177 Both of these steroids are additionally difficult to measure by LC-MS/MS because of their preference to ionize in negative ion mode which is typically less sensitive than positive ion mode. Nevertheless, they can still be measured using high-grade mass spectrometer instruments and careful attention to sample preparation. The use of LC-MS/MS in the field of endocrinology is not restricted to the measurement of steroids and has been applied to other analytes which have proved difficult to measure by alternative means such as urine^178–180 and plasma^181–185 metadrenalines, including measurement of 3-methoxytyramine (3-MT). 3-MT is not measured by immunoassay but is useful for detecting the likelihood of metastasis in paragangliomas.¹⁸⁶ Other applications include plasma renin activity^187,188 and in a reference method capacity for, e.g. thyroxine.^56,58,62

Paediatrics

MS has been a feature in paediatric laboratories for many years. It is used for the measurement of dried blood spot acylcarnitines for newborn medium chain acyl coA dehydrogenase deficiency (MCADD)^189,190 and phenylalanine and tyrosine for phenylketonuria screening.⁵¹ An extended newborn screening pilot has recently been conducted which would include tests for five additional metabolic conditions in the same analysis as the existing screening process. These five additional conditions are maple syrup urine disease, homocystinuria, glutaric acidaemia type 1, long-chain acyl coA dehydrogenase deficiency and isovaleric acidaemia.¹⁹¹

The use of MS for the investigation of inborn errors of metabolism typically uses an alternative approach to quantitative assays. For example, acyl carnitines are derivatized to form their butyl esters.¹⁹⁰ Once derivatized, all acyl carnitines give a product ion with a mass-to-charge ratio (m/z) of 85. A precursor scan is performed of all m/z that give a product of 85, and the output is a scan rather than a chromatogram. An elevated acylcarnitine can be identified by its m/z. However, the newborn screening programme for MCADD may use a more targeted approach, for just C8 and C10 acylcarnitines with or without derivitization.^190,192

Neutral loss scans of derivatized amino acids can be performed in the investigation of amino acid disorders. In this case, all parent and daughter ions are scanned with a specific loss.^49,51,193 The loss is m/z 102 for acidic and neutral amino acids and m/z 119 for basic amino acids. Alternatively, non-derivatized methods are available for phenylalanine and tyrosine with the loss of formate during the CID stage and may be used for newborn screening.¹⁹⁴ The use of scan mode rather than MRM mode for acylcarnitines and amino acids means that only one analysis needs to be performed to screen for many related disorders with quicker run-times than gas chromatography-based assays.

Quantitative LC-MS/MS also plays a role in paediatrics. After the diagnosis of conditions by screening techniques, quantitative assays are used for disease monitoring.¹⁹⁵ Specific quantitative assays may also be used in the investigation of disorders such as those of bile acid synthesis.¹⁹⁶ Hexanoylglycine may also be measured in the investigation of fatty acid disorders.¹⁹⁷

Steroid assays are applicable to the paediatric population due to multiplexing resulting in reduced overall sample volume and the ability to measure uncommon steroids.¹⁹⁸ There are methods in the literature for urine steroid measurement by LC-MS/MS for the diagnosis of congenital adrenal hyperplasia (CAH) with a reduced false-positive rate;¹⁹⁹ however, this still typically remains a GC-MS assay. Second-tier testing for CAH is possible by the measurement of 17-hydroxyprogesterone or 11-deoxycortisol in plasma.^{48,160,199,200}

Small molecule quantitation

The availability of LC-MS/MS in the clinical laboratory for large volume tests such as immunosuppressants, vitamin D or newborn screening gives the user the flexibility to consolidate a range of esoteric or very specialist assays for very little additional cost. Where separate assays exist such as high-pressure liquid chromatography (HPLC), enzyme-linked immunosorbent assay or colourimetric methods, additional equipment is required. These may include HPLC equipment and detectors, plate washer and plate readers and spectrophotometers and perhaps also reagent kits. Usually when these assays are converted to LC-MS/MS, additional cost is minimal, the main outlay being the analytical column and internal standard. This may be the reason why there are also niche small molecule assays reported in the literature that are performed by LC-MS/MS such as urine oxalate,^73,75,201 citrate²⁰² and cystine^69,78 which are used in renal stone clinics. Urine^37,77,81 and/or plasma^16,74,203 5-hydroxyindolacetic acid may be measured for the investigation or monitoring of neuroendocrine tumours. Markers of nicotine use such as cotinine^204–208 may be used in patients with lung disease or transplants to monitor abstinence.

Microbiology

Quantitative LC-MS/MS has an established role in the measurement of antibiotic^{79,111,112,209} or antifungal^113–116 drugs in Biochemistry, Microbiology or Mycology Departments. However, qualitative MS has an emerging role in the routine Microbiology Department in the form of matrix-assisted laser desorption ionization time of flight (MALDI-TOF) MS. Using this technique, an isolated colony is smeared onto the sampling slide before presentation to the MALDI-TOF instrument. After application of a 337-nm ultraviolet nitrogen laser, a collection of ionized peptides, specific to the organism, enter the flight tube. A spectrum is produced which can be compared to a library to identify the organism responsible for the colony. MALDI-TOF is reported to be up to 24 h faster at species diagnosis than typical microbiological or molecular techniques^{70,80,210,211} and has a high sample throughput. However, this technique has been shown not to be able to differentiate between closely related bacterial species such as Escherichia Coli and Shigella Sp due to similar peptide content,^212,213 and other techniques such as culture, staining and phenotyping may still be required.

Current analytical considerations

Validation

As with all in-house assays, LC-MS/MS assays must be subject to thorough method validation as described in more detail by Honour²¹⁴ in 2011. Other guidelines for method validation have been quoted in the literature, e.g. the ACB guidelines,²¹⁵ clinical laboratory improvement amendments,²¹⁶ European medicines agency^217,218 and the food and drug administration.²¹⁹ Local guidelines that laboratories use may soon require a revision if they are to meet ISO 15189 criteria. A particular area for consideration for in-house LC-MS/MS assays will be the monitoring and assessment of lot numbers and batches of reagents as these may be made frequently.

Batch versus random access

Typically, quantitative MS assays are performed in the clinical laboratory as batches rather than random access as in automated immunoassays. This type of analysis limits MS to those analytes for which a rapid turnaround time is not required. Automated sample handling coupled to mass spectrometers may go some way to reduce variability and thus the possibility of a more flexible approach, and such systems have been described for use in the microbiology laboratory²¹⁰ and for urine drug analysis.²⁰² Although such systems may not necessarily decrease the time required for sample preparation and analysis, they do decrease the amount of hands-on time required, which is a major advantage in today’s busy clinical laboratory.

A novel technique termed isotopic internal calibration (ICAL) utilizes differing concentrations of various stable isotopes that are added to samples to produce a calibration within the sample therefore not requiring additional time to analyse a multipoint calibration curve.²⁰¹ This could offer the ability to prepare and analyse samples in a random access fashion if required. This technique may still require significant staff time to prepare the sample and ensure the correct mobile phases and analytical column is in place if this differs from what had run previously. However, this is an advance towards random access MS.

Emerging and future applications

Automated sample handling

A major limitation to the use of quantitative MS in the routine clinical laboratory is limited throughput due to time-consuming manual sample preparation which requires skilled operator time.^220,221 Many laboratories have invested in automated sample handling techniques to reduce the time needed.^{92,105,115,202,222–224} Methods described using automated sample handling robots often describe tube identification using barcodes, sample and reagent pipetting and may even incorporate sample extraction such as SPE.^105,202 These methods often still require operator input such as mixing, drying, transferring and/or centrifugation steps.¹⁰⁵ Extractions involving solvents are also possible;¹⁴¹ however, it is often difficult to accommodate these instruments within extraction units.

An attractive innovation would be an extraction technique that can be automated without requiring operator input from when the samples and reagents are loaded until the extracts are prepared. The direct extraction-elution method is an example of sample extraction that does not require precipitation, filtration, centrifugation or solvent evaporation²²⁵ but is limited to neutral molecules such as vitamin D.

An alternative method of simplified, automated sample extraction utilizes magnetic microparticles with a functionalized surface to attract the analyte of interest while allowing the sample matrix to be removed from the analytical system.^226,227 This approach has been successfully applied to a mycophenolic acid assay.²²⁷

Pay-per-sample

The introduction of MS into the laboratory usually follows a capital purchase or a fixed term leasing arrangement. A novel approach provided by a collaboration of Anatune (Friiscan) and Agilent Technologies provides mass spectrometers with integrated sample preparation and preprepared methods that are run on a cost-per sample basis. This approach does not require the expertise in method development and maintenance as methods are supplied and supported by the manufacturer. Methods available include vitamin D,²²⁸ immunosuppressants²²⁹ and some steroids.²³⁰ However, the use of the equipment is restricted to these applications, and they may be more expensive than in-house alternatives.

Peptide and protein quantitation

MS has been used as a tool in the proteomics field for many years particularly for protein identification using MALDI-TOF.^231–233 Protein and peptide quantitation using tandem MS is a more recent advance.^234–237 Tandem mass spectrometers have a limit to the size of molecule that can be quantified, often around a mass to charge ratio of 2000 Daltons. Peptides with a mass greater than this upper limit can still be quantified if they carry multiple charges;²³⁸ however, larger peptides and proteins must be digested prior to the measurement of smaller peptides.^239–241 Analogue proteins²⁴² or isotope-labelled proteins²⁴³ as internal standards may be added to the sample prior to digestion or labelled peptides can be added later in the process.^{239,240,244,245}

Using MS for protein measurement may address issues that affect the current methodology such as immunoassays for thyroglobulin. Thyroglobulin immunoassays are renowned for having interferences from heterophilic antibodies, and thyroglobulin measurement is a challenge due to its low abundance in serum.^239,240 One reported assay combines immunoaffinity peptide enrichment and tryptic digest for the preparation of peptides.²⁴⁶ Trypsin also was noted to digest the immunoglobulins that potentially interfere in the immunoassays, and MS allows three peptides to be monitored simultaneously.²⁴⁶

Quantitation of growth hormone has also been of interest. It consists of several isoforms and variants due to post-translational modifications and complex formation. In these assays, key peptides are selected which are believed to be representative of the measurand;²⁴⁷ however, such assays may not have sufficient sensitivity for all applications, such as detecting growth hormone deficiency.

A novel analytical technique, mass spectrometric immunoassay (MSIA) has been described for serum and plasma protein analysis^248–250 (Figure 5). These assays utilize antibody-coated pipette tips to prepare samples and enrich proteins and peptides prior to analysis. These assays are reported to be rapid and high throughput for the analysis of e.g. parathyroid hormone (PTH)²⁴⁹ and prostate-specific antigen.²⁵⁰

Figure 5.

MSIA. Antibodies are bound to a tip which is loaded with sample. The analyte of interest will bind the antibody. Wash steps are performed to remove unbound compounds followed by buffer elution to remove the analyte of interest into a collection plate for further sample preparation.

Metabolomics

Metabolomics is a key area of biomarker discovery where the global metabolite profile of test groups is compared to control groups.²⁵¹ GC-MS has typically been used in metabolomic laboratories^252,253 but is limited to volatile and derivatizable compounds. LC-MS is increasingly being used as a complementary technique due to its ability to analyse a wider range of chemical species;^254–257 however, top-end instruments including TOF capability are often required for biomarker identification.²⁵⁴ Once biomarkers are identified, LC-MS/MS can be employed due to the rapid scan times of newer instruments to quantify large panels of metabolites simultaneously.²⁵⁸

Point of care testing

MS has recently hit the headlines for its use in the operating theatre as a point of care testing device at Imperial College London. In this case, a technique called rapid evaporative ionization MS is used.^259–262 The surgeon’s knife is connected to a mass spectrometer, and as the surgeon cuts through the tissue, some is vaporized and passed to the MS to produce spectra which can differentiate between healthy and cancerous tissue by comparison to a reference library. An initial study of 91 patients undergoing surgery has had a 100% success rate of determining whether the samples were cancerous or not which in the future may remove the need for intraoperative histology.²⁵⁹ This advance shows that MS has an emerging role outside the laboratory and in direct clinical areas.

Total laboratory automation and random access

A combination of factors already described such as ICAL,²⁰¹ automated and/or simplified sample preparation,^{92,105,115,202,222–227} manufacturer prepared calibrators and reagents^30–33 has the potential, if combined, to offer a completely random access solution to MS. If this is further combined with laboratory preanalytics such as centrifuges, decappers and tracking systems, a total laboratory automation solution could be a reality.

Conclusion

MS has many applications in the clinical laboratory, and the above is by no means an exhaustive list. Despite being considered to be a technically demanding process, manufacturers are making efforts to simplify sample handling, and many laboratories have adopted MS on the back of kit methods that are increasingly available. One of the main disadvantages is that assays are typically batched, and random access has not been universally developed; however, we envisage that in the coming years, further significant advancements in this technology will enable MS to be as easily used as main clinical chemistry analysers.

Footnotes

Acknowledgements

This article was prepared at the invitation of the Clinical Sciences Reviews Committee of the Association for Clinical Biochemistry and Laboratory Medicine.

Declaration of conflicting interests

None declared.

Funding

This review received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Ethical approval

Not applicable.

Guarantor

LJO.

Contributorship

LJO and JEA researched, wrote and edited the manuscript. BGK reviewed and edited the manuscript.

References

Whitehouse

Dreyer

Yamashita

. Electrospray interface for liquid chromatographs and mass spectrometers. Anal Chem 1985; 57: 675–679.

Horning

Carroll

Dzidic

. Atmospheric pressure ionization (API) mass spectrometry. Solvent-mediated ionization of samples introduced in solution and in a liquid chromatograph effluent stream. J Chromatogr Sci 1974; 12: 725–729.

Nguyen

Fenn

. Gas-phase ions of solute species from charged droplets of solutions. Proc Natl Acad Sci U S A 2007; 104: 1111–1117.

Dole

Mack

Hines

. Molecular beams of macroions. J Chem Phys 1968; 49: 2240–2248.

de Hoffman

. Tandem mass spectrometry: a primer. J Mass Spectrom 1996; 31: 129–137.

Keevil

McCann

Cooper

. Evaluation of a rapid micro-scale assay for tacrolimus by liquid chromatography-tandem mass spectrometry. Ann Clin Biochem 2002; 39: 487–492.

Keevil

Tierney

Cooper

. Simultaneous and rapid analysis of cyclosporin A and creatinine in finger prick blood samples using liquid chromatography tandem mass spectrometry and its application in C2 monitoring. Ther Drug Monit 2002; 24: 757–767.

Annesley

Clayton

. Simple extraction protocol for analysis of immunosuppressant drugs in whole blood. Clin Chem 2004; 50: 1845–1848.

Ceglarek

Lembcke

Fiedler

. Rapid simultaneous quantification of immunosuppressants in transplant patients by turbulent flow chromatography combined with tandem mass spectrometry. Clin Chim Acta 2004; 346: 181–190.

10.

Deters

Kirchner

Resch

. Simultaneous quantification of sirolimus, everolimus, tacrolimus and cyclosporine by liquid chromatography-mass spectrometry (LC-MS). Clin Chem Lab Med 2002; 40: 285–292.

11.

Koseki

Nakashima

Nagae

. Simultaneous quantitative determination of cyclosporine A and its three main metabolites (AM1, AM4N and AM9) in human blood by liquid chromatography/mass spectrometry using a rapid sample processing method. Rapid Commun Mass Spectrom 2006; 20: 733–740.

12.

Taylor

Jones

Martin

. Microscale high-performance liquid chromatography-electrospray tandem mass spectrometry assay for cyclosporin A in blood. J Chromatogr B Biomed Sci Appl 1998; 705: 289–294.

13.

Vidal

Kirchner

Wunsch

. Automated simultaneous quantification of the immunosuppressants 40-O-(2-hydroxyethyl) rapamycin and cyclosporine in blood with electrospray-mass spectrometric detection. Clin Chem 1998; 44: 1275–1282.

14.

Biotage. Extraction of a range of immunosuppressants from whole blood using ISOLUTE® SLE+ prior to LC-MS/MS analysis 2010. Biotage, http://www.biotage.com/literature/download/an758_extraction_of_a_range_of_immunosuppressants_from_whole_blood_sle.pdf?ref=http%3A%2F%2Fwww.biotage.com%2Fliterature%2Fsearch%3Fkeywords%255B%255D%255B%255D%3D102 (2013, accessed 14 July 2014).

15.

Patel

Sharma

Sanyal

. Analysis of second-generation antidepressant drug, sertraline and its active metabolite, N-desmethyl sertraline in human plasma by a sensitive and selective liquid chromatography-tandem mass spectrometry method. J Chromatogr B Anal Technol Biomed Life Sci 2009; 877: 221–229.

16.

Keevil

Macdonald

Macdowall

. Salivary testosterone measurement by liquid chromatography tandem mass spectrometry in adult males and females. Ann Clin Biochem 2013; 51: 368–378.

17.

Gallagher

Owen

Keevil

. Simultaneous determination of androstenedione and testosterone in human serum by liquid chromatography-tandem mass spectrometry. Ann Clin Biochem 2007; 44: 48–56.

18.

Chen

Liu

Zhong

. Trimethylsilyldiazomethane derivatization coupled with solid-phase extraction for the determination of alendronate in human plasma by LC-MS/MS. Anal Bioanal Chem 2012; 402: 791–798.

19.

Harder

Koletzko

Peissner

. Quantification of 22 plasma amino acids combining derivatization and ion-pair LC-MS/MS. J Chromatogr B Anal Technol Biomed Life Sci 2011; 879: 495–504.

20.

Ramos

. Critical overview of sample preparation techniques. J Chromatogr A 2012; 1221: 1221–1236.

21.

Carter

Jones

. Use of a common standard improves the performance of liquid chromatography-tandem mass spectrometry methods for serum 25-hydroxyvitamin-D. Ann Clin Biochem 2009; 46: 79–81.

22.

Yates

Bowron

Calton

. Interlaboratory variation in 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 is significantly improved if common calibration material is used. Clin Chem 2008; 54: 2082–2084.

23.

Maunsell

Wright

Rainbow

. Routine isotope-dilution liquid chromatography-tandem mass spectrometry assay for simultaneous measurement of the 25-hydroxy metabolites of vitamins D2 and D3. Clin Chem 2005; 51: 1683–1690.

24.

Owen

Adaway

Davies

. Development of a rapid assay for the analysis of serum cortisol and its implementation into a routine service laboratory. Ann Clin Biochem 2013; 50: 345–352.

25.

Keevil

Newman

Lockhart

. Validation of an assay for voriconazole in serum samples using liquid chromatography-tandem mass spectrometry. Ther Drug Monit 2004; 26: 650–657.

26.

Fisher

Handley

Taylor

. Measurement of quetiapine and four quetiapine metabolites in human plasma by LC-MS/MS. Biomed Chromatogr BMC 2012; 26: 1125–1132.

27.

Cawood

Field

Ford

. Testosterone measurement by isotope-dilution liquid chromatography-tandem mass spectrometry: validation of a method for routine clinical practice. Clin Chem 2005; 51: 1472–1479.

28.

Nguyen

Nethrapalli

. Evaluation of matrix effects in analysis of estrogen using liquid chromatography-tandem mass spectrometry. J Sep Sci 2011; 34: 1781–1787.

29.

Hewavitharana

. Matrix matching in liquid chromatography-mass spectrometry with stable isotope labelled internal standards–is it necessary? J Chromatogr A 2011; 1218: 359–361.

30.

Guilhaumou

Lacarelle

Sampol-Manos

. A rapid, simple and sensitive liquid chromatography-tandem mass spectrometry method for routine clinical monitoring of tacrolimus with the Waters Masstrak immmunosuppressant kit. Methods Find Exp Clin Pharmacol 2010; 32: 737–743.

31.

Kim

Chung

. Evaluation of the masstrak immunosuppressant XE kit for the determination of everolimus and cyclosporin A in human whole blood employing isotopically labeled internal standards. Clin Chem Lab Med 2011; 49: 2021–2027.

32.

Koal

Schmiederer

Pham-Tuan

. Standardized LC-MS/MS based steroid hormone profile-analysis. J Steroid Biochem Mol Biol 2012; 129: 129–138.

33.

Gervasoni

Cocci

Zuppi

. Total 25-hydroxyvitamin D determination by an entry level triple quadrupole instrument: comparison between two commercial kits. BioMed Res Int 2013; 2013: 270426–270426.

34.

Keevil

. Novel liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for measuring steroids. Best Pract Res Clin Endocrinol Metabol 2013; 27: 663–674.

35.

Annesley

. Ion suppression in mass spectrometry. Clin Chem 2003; 49: 1041–1044.

36.

Matuszewski

Constanzer

Chavez-Eng

. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem 2003; 75: 3019–3030.

37.

Berg

Strand

. (1)(3)C labelled internal standards–a solution to minimize ion suppression effects in liquid chromatography-tandem mass spectrometry analyses of drugs in biological samples? J Chromatogr A 2011; 1218: 9366–9374.

38.

Ferreiros

Dresen

Alonso

. Validated quantitation of angiotensin II receptor antagonists (ARA-II) in human plasma by liquid-chromatography-tandem mass spectrometry using minimum sample clean-up and investigation of ion suppression. Ther Drug Monit 2007; 29: 824–834.

39.

Bunch

El-Khoury

Gabler

. Do deuterium labeled internal standards correct for matrix effects in LC-MS/MS assays? A case study using plasma free metanephrine and normetanephrine. Clin Chim Acta 2014; 429: 4–5.

40.

Mallet

Mazzeo

. A study of ion suppression effects in electrospray ionization from mobile phase additives and solid-phase extracts. Rapid Commun Mass Spectrom 2004; 18: 49–58.

41.

Chocholous

Kosarova

Satinsky

. Enhanced capabilities of separation in sequential injection chromatography–fused-core particle column and its comparison with narrow-bore monolithic column. Talanta 2011; 85: 1129–1134.

42.

Couchman

Benton

Moniz

. Variability in the analysis of 25-hydroxyvitamin D by liquid chromatography-tandem mass spectrometry: the devil is in the detail. Clin Chim Acta 2012; 413: 1239–1243.

43.

Owen

Macdonald

Keevil

. Is calibration the cause of variation in liquid chromatography tandem mass spectrometry testosterone measurement? Ann Clin Biochem 2013; 50: 368–370.

44.

Granado Lorencio

Blanco-Navarro

Perez-Sacrsitan

. Critical evaluation of assays for vitamin D status. Curr Opin Clin Nutr Metabol Care 2013; 16: 734–740.

45.

Bowers

Jr Fassett

White

. Isotope dilution mass spectrometry and the national reference system. Anal Chem 1993; 65: 475r–479r.

46.

Inghram

. Stable isotope dilution as an analytical tool. Annu Rev Nucl Sci 1954; 4: 81–92.

47.

Kushnir

Blamires

Rockwood

. Liquid chromatography-tandem mass spectrometry assay for androstenedione, dehydroepiandrosterone, and testosterone with pediatric and adult reference intervals. Clin Chem 2010; 56: 1138–1147.

48.

Rauh

Gröschl

Rascher

. Automated, fast and sensitive quantification of 17 alpha-hydroxy-progesterone, androstenedione and testosterone by tandem mass spectrometry with on-line extraction. Steroids 2006; 71: 450–458.

49.

Chace

Hillman

Millington

. Rapid diagnosis of maple syrup urine disease in blood spots from newborns by tandem mass spectrometry. Clin Chem 1995; 41: 62–68.

50.

Chace

Hillman

Millington

. Rapid diagnosis of homocystinuria and other hypermethioninemias from newborns' blood spots by tandem mass spectrometry. Clin Chem 1996; 42: 349–355.

51.

Chace

Millington

Terada

. Rapid diagnosis of phenylketonuria by quantitative analysis for phenylalanine and tyrosine in neonatal blood spots by tandem mass spectrometry. Clin Chem 1993; 39: 66–71.

52.

Kaiser

Akerboom

Molnar

. Modified HPLC-electrospray ionization/mass spectrometry method for HbA1c based on IFCC reference measurement procedure. Clin Chem 2008; 54: 1018–1022.

53.

Kaiser

Akerboom

Ohlendorf

. Liquid chromatography-isotope dilution-mass spectrometry as a new basis for the reference measurement procedure for hemoglobin A1c determination. Clin Chem 2010; 56: 750–754.

54.

Liebisch

Binder

Schifferer

. High throughput quantification of cholesterol and cholesteryl ester by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Biochim Biophys Acta 2006; 1761: 121–128.

55.

Zhou

Dong

. Serum cholesterol measured by isotope dilution liquid chromatography tandem mass spectrometry. Clin Chem Lab Med 2011; 49: 669–676.

56.

Ackermans

Kettelarij-Haas

Boelen

. Determination of thyroid hormones and their metabolites in tissue using SPE UPLC-tandem MS. Biomed Chromatogr 2012; 26: 485–490.

57.

Gounden

Jonklaas

Soldin

. A pilot study: subclinical hypothyroidism and free thyroid hormone measurement by immunoassay and mass spectrometry. Clin Chim Acta 2014; 430: 121–124.

58.

Jonklaas

Kahric-Janicic

Soldin

. Correlations of free thyroid hormones measured by tandem mass spectrometry and immunoassay with thyroid-stimulating hormone across 4 patient populations. Clin Chem 2009; 55: 1380–1388.

59.

Soldin

. Thyroid hormone testing by tandem mass spectrometry. Clin Biochem 2011; 44: 89–94.

60.

van Deventer

Soldin

. The expanding role of tandem mass spectrometry in optimizing diagnosis and treatment of thyroid disease. Adv Clin Chem 2013; 61: 127–152.

61.

Wang

Stapleton

. Analysis of thyroid hormones in serum by liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem 2010; 397: 1831–1839.

62.

Yue

Rockwood

Sandrock

. Free thyroid hormones in serum by direct equilibrium dialysis and online solid-phase extraction–liquid chromatography/tandem mass spectrometry. Clin Chem 2008; 54: 642–651.

63.

Ansermot

Fathi

Veuthey

. Quantification of cyclosporine and tacrolimus in whole blood. Comparison of liquid chromatography-electrospray mass spectrometry with the enzyme multiplied immunoassay technique. Clin Biochem 2008; 41: 910–913.

64.

Keevil

Tierney

Cooper

. Rapid liquid chromatography-tandem mass spectrometry method for routine analysis of cyclosporin A over an extended concentration range. Clin Chem 2002; 48: 69–76.

65.

Meinitzer

Gartner

Pilz

. Ultra fast liquid chromatography-tandem mass spectrometry routine method for simultaneous determination of cyclosporin A, tacrolimus, sirolimus, and everolimus in whole blood using deuterated internal standards for cyclosporin A and everolimus. Ther Drug Monit 2010; 32: 61–66.

66.

Buchwald

Winkler

Epting

. Validation of an LC-MS/MS method to determine five immunosuppressants with deuterated internal standards including MPA. BMC Clin Pharmacol 2012; 12: 1–11.

67.

Wiegand

Lorusso

. A stable isotope-labeled internal standard is essential for correcting for the interindividual variability in the recovery of lapatinib from cancer patient plasma in quantitative LC-MS/MS analysis. J Chromatogr B Analyt Technol Biomed Life Sci 2013; 941: 100–108.

68.

Stokvis

Rosing

Lopez-Lazaro

. Switching from an analogous to a stable isotopically labeled internal standard for the LC-MS/MS quantitation of the novel anticancer drug Kahalalide F significantly improves assay performance. Biomed Chromatogr BMC 2004; 18: 400–402.

69.

Chavez-Eng

Constanzer

Matuszewski

. High-performance liquid chromatographic-tandem mass spectrometric evaluation and determination of stable isotope labeled analogs of rofecoxib in human plasma samples from oral bioavailability studies. J Chromatogr B Anal Technol Biomed Life Sci 2002; 767: 117–129.

70.

Davison

Milan

Dutton

. Potential problems with using deuterated internal standards for liquid chromatography-tandem mass spectrometry. Ann Clin Biochem 2013; 50: 274–274.

71.

Owen

Adaway

Morris

. A widely applicable plasma renin activity assay by LC-MS/MS with offline solid phase extraction. Ann Clin Biochem 2014; 51: 409–411.

72.

Carter

Owen

Kerstens

. A liquid chromatography tandem mass spectrometry assay for plasma renin activity using online solid-phase extraction. Ann Clin Biochem 2012; 49: 570–579.

73.

Duxbury

Owen

Gillingwater

. Naturally occurring isotopes of an analyte can interfere with doubly deuterated internal standard measurement. Ann Clin Biochem 2008; 45: 210–212.

74.

Cheze

Deveaux

Martin

. Simultaneous analysis of six amphetamines and analogues in hair, blood and urine by LC-ESI-MS/MS. Application to the determination of MDMA after low ecstasy intake. Forensic Sci Int 2007; 170: 100–104.

75.

Cheze

Villain

Pepin

. Determination of bromazepam, clonazepam and metabolites after a single intake in urine and hair by LC-MS/MS. Application to forensic cases of drug facilitated crimes. Forensic Sci Int 2004; 145: 123–130.

76.

Gosetti

Mazzucco

Gennaro

. Ultra high performance liquid chromatography tandem mass spectrometry determination and profiling of prohibited steroids in human biological matrices. A review. J Chromatogr B Anal Technol Biomed Life Sci 2013; 927: 22–36.

77.

Villain

Concheiro

Cirimele

. Screening method for benzodiazepines and hypnotics in hair at pg/mg level by liquid chromatography-mass spectrometry/mass spectrometry. J Chromatogr B Anal Technol Biomed Life Sci 2005; 825: 72–78.

78.

Jones

Owen

Adaway

. Simultaneous analysis of cortisol and cortisone in saliva using XLC-MS/MS for fully automated online solid phase extraction. J Chromatogr B Anal Technol Biomed Life Sci 2012; 881–882: 42–48.

79.

Owen

Haslam

Adaway

. A simplified liquid chromatography tandem mass spectrometry assay, using on-line solid-phase extraction, for the quantitation of cortisol in saliva and comparison with a routine DELFIA method. Ann Clin Biochem 2010; 47: 131–136.

80.

Bright

Frick

Out

. Individual differences in the cortisol and salivary alpha-amylase awakening responses in early childhood: relations to age, sex, and sleep. Dev Psychobiol 2014; 56: 1300–1315.

81.

Elias

Martinez

Barone

. Late-night salivary cortisol has a better performance than urinary free cortisol in the diagnosis of Cushing's syndrome. J Clin Endocrinol Metab 2014; 99: 2045–2051.

82.

Frost

Bellingham

. Neonatal haemoglobinopathy screening. Acta Haematol 1987; 78: 142–143.

83.

Guthrie

Susi

. A simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants. Pediatrics 1963; 32: 338–343.

84.

Wallace

Beastall

Cook

. Neonatal screening for congenital adrenal hyperplasia: a programme based on a novel direct radioimmunoassay for 17-hydroxyprogesterone in blood spots. J Endocrinol 1986; 108: 299–308.

85.

Keevil

. The analysis of dried blood spot samples using liquid chromatography tandem mass spectrometry. Clin Biochem 2011; 44: 110–118.

86.

van den Bosch

Oemardien

Srebniak

. Prenatal screening of sialic acid storage disease and confirmation in cultured fibroblasts by LC-MS/MS. J Inherit Metab Dis 2011; 34: 1069–1073.

87.

Srinivas

Mullangi

. An overview of various validated HPLC and LC-MS/MS methods for quantitation of drugs in bile: challenges and considerations. Biomed Chromatogr 2011; 25: 65–81.

88.

Mora

Ceglarek

Manzotti

. Cyclosporin A in the ocular fluids of uveitis patients following long-term systemic administration. Graefe's Arch Clin Exp Ophthalmol 2008; 246: 1047–1052.

89.

Oeckl

Ferger

. Simultaneous LC-MS/MS analysis of the biomarkers cAMP and cGMP in plasma, CSF and brain tissue. J Neurosci Methods 2012; 203: 338–343.

90.

Wong

Zuo

. Investigation of the disposition of loxapine, amoxapine and their hydroxylated metabolites in different brain regions, CSF and plasma of rat by LC-MS/MS. J Pharm Biomed Anal 2012; 58: 83–93.

91.

Annesley

McKeown

Holt

. Standardization of LC-MS for therapeutic drug monitoring of tacrolimus. Clin Chem 2013; 59: 1630–1637.

92.

Christians

Jacobsen

Serkova

. Automated, fast and sensitive quantification of drugs in blood by liquid chromatography-mass spectrometry with on-line extraction: immunosuppressants. J Chromatogr B Biomed Sci Appl 2000; 748: 41–53.

93.

Ansermot

Fathi

Veuthey

. Simultaneous quantification of cyclosporine, tacrolimus, sirolimus and everolimus in whole blood by liquid chromatography-electrospray mass spectrometry. Clin Biochem 2008; 41: 728–735.

94.

Ceglarek

Casetta

Lembcke

. Inclusion of MPA and in a rapid multi-drug LC-tandem mass spectrometric method for simultaneous determination of immunosuppressants. Clin Chim Acta 2006; 373: 168–171.

95.

Keevil

Fildes

Baynes

. Liquid chromatography-mass spectrometry measurement of tacrolimus in finger-prick samples compared with venous whole blood samples. Ann Clin Biochem 2009; 46: 144–145.

96.

Acott

. Home fingerprick sampling for immunosuppressant drug monitoring in pediatric renal transplant patients. Nat Clin Pract Nephrol 2006; 2: 304–305.

97.

Yonan

Martyszczuk

Machaal

. Monitoring of cyclosporine levels in transplant recipients using self-administered fingerprick sampling. Clin Transplant 2006; 20: 221–225.

98.

Webb

Roberts

Preziosi

. Fingerprick blood samples can be used to accurately measure tacrolimus levels by tandem mass spectrometry. Pediatr Transplant 2005; 9: 729–733.

99.

Mee

Wong

Sun

. Monitoring of cyclosporine concentrations by using dry blood-spot samples. J Clin Lab Anal 1991; 5: 74–77.

100.

den Burger

Wilhelm

Chahbouni

. Analysis of cyclosporin A, tacrolimus, sirolimus, and everolimus in dried blood spot samples using liquid chromatography tandem mass spectrometry. Anal Bioanal Chem 2012; 404: 1803–1811.

101.

Hoogtanders

van der Heijden

Christiaans

. Therapeutic drug monitoring of tacrolimus with the dried blood spot method. J Pharm Biomed Anal 2007; 44: 658–664.

102.

van der Heijden

de Beer

Hoogtanders

. Therapeutic drug monitoring of everolimus using the dried blood spot method in combination with liquid chromatography-mass spectrometry. J Pharm Biomed Anal 2009; 50: 664–670.

103.

Cao

Huang

. Development and validation of a sensitive LC-MS/MS method for determination of tacrolimus on dried blood spots. Biomed Chromatogr 2013; 27: 327–334.

104.

Hinchliffe

Adaway

Keevil

. Simultaneous measurement of cyclosporin A and tacrolimus from dried blood spots by ultra high performance liquid chromatography tandem mass spectrometry. J Chromatogr B Anal Technol Biomed Life Sci 2012; 883–884: 102–107.

105.

McMahon

Luo

Hayes

. High-throughput analysis of everolimus (RAD001) and cyclosporin A (CsA) in whole blood by liquid chromatography/mass spectrometry using a semi-automated 96-well solid-phase extraction system. Rapid Commun Mass Spectrom 2000; 14: 1965–1971.

106.

Bardin

Ottinger

Breau

. Determination of free levels of phenytoin in human plasma by liquid chromatography/tandem mass spectrometry. J Pharm Biomed Anal 2000; 23: 573–579.

107.

Beck

Ohman

Nordgren

. Determination of lamotrigine and its metabolites in human plasma by liquid chromatography-mass spectrometry. Ther Drug Monit 2006; 28: 603–607.

108.

Blonk

van der Nagel

Smit

. Quantification of levetiracetam in plasma of neonates by ultra performance liquid chromatography-tandem mass spectrometry. J Chromatogr B Anal Technol Biomed Life Sci 2010; 878: 675–681.

109.

Breton

Cociglio

Bressolle

. Liquid chromatography-electrospray mass spectrometry determination of carbamazepine, oxcarbazepine and eight of their metabolites in human plasma. J Chromatogr B Anal Technol Biomed Life Sci 2005; 828: 80–90.

110.

Jansson

Karvanen

Cars

. Quantitative analysis of colistin A and colistin B in plasma and culture medium using a simple precipitation step followed by LC/MS/MS. J Pharm Biomed Anal 2009; 49: 760–767.

111.

Chen

Liang

Chen

. Quantitative determination of azithromycin in human plasma by liquid chromatography-mass spectrometry and its application in a bioequivalence study. J Pharm Biomed Anal 2006; 42: 480–487.

112.

Wang

Sun

. Simultaneous determination of erythromycin ethylsuccinate and its metabolite erythromycin in human plasma using liquid chromatography-electrospray ionization mass spectrometry for clinical study. J Pharm Biomed Anal 2006; 40: 737–743.

113.

Baietto

D'Avolio

Ventimiglia

. Development, validation, and routine application of a high-performance liquid chromatography method coupled with a single mass detector for quantification of itraconazole, voriconazole, and posaconazole in human plasma. Antimicrob Agents Chemother 2010; 54: 3408–3413.

114.

Decosterd

Rochat

Pesse

. Multiplex ultra-performance liquid chromatography-tandem mass spectrometry method for simultaneous quantification in human plasma of fluconazole, itraconazole, hydroxyitraconazole, posaconazole, voriconazole, voriconazole-N-oxide, anidulafungin, and caspofungin. Antimicrob Agents Chemother 2010; 54: 5303–5315.

115.

Egle

Trittler

Konig

. Fast, fully automated analysis of voriconazole from serum by LC-LC-ESI-MS-MS with parallel column-switching technique. J Chromatogr B Anal Technol Biomed Life Sci 2005; 814: 361–367.

116.

Farowski

Cornely

Vehreschild

. Quantitation of azoles and echinocandins in compartments of peripheral blood by liquid chromatography-tandem mass spectrometry. Antimicrob Agents Chemother 2010; 54: 1815–1819.

117.

Adaway

Keevil

. Therapeutic drug monitoring and LC-MS/MS. J Chromatogr B Anal Technol Biomed Life Sci 2012; 883–884: 33–49.

118.

Aouri

Moradpour

Cavassini

. Multiplex liquid chromatography-tandem mass spectrometry assay for simultaneous therapeutic drug monitoring of ribavirin, boceprevir, and telaprevir. Antimicrob Agents Chemother 2013; 57: 3147–3158.

119.

Danso

Langman

Snozek

. LC-MS/MS quantitation of ribavirin in serum and identification of endogenous isobaric interferences. Clin Chim Acta 2011; 412: 2332–2335.

120.

Perogamvros

Owen

Newell-Price

. Simultaneous measurement of cortisol and cortisone in human saliva using liquid chromatography-tandem mass spectrometry: application in basal and stimulated conditions. J Chromatogr B Anal Technol Biomed Life Sci 2009; 877: 3771–3775.

121.

Aravagiri

Marder

. Simultaneous determination of clozapine and its N-desmethyl and N-oxide metabolites in plasma by liquid chromatography/electrospray tandem mass spectrometry and its application to plasma level monitoring in schizophrenic patients. J Pharm Biomed Anal 2001; 26: 301–311.

122.

Ming

Heathcote

. Therapeutic drug monitoring of clozapine and norclozapine in human serum using ultra-performance liquid chromatography- tandem mass spectrometry. J Anal Toxicol 2009; 33: 198–203.

123.

Niederlander

Koster

Hilhorst

. High throughput therapeutic drug monitoring of clozapine and metabolites in serum by on-line coupling of solid phase extraction with liquid chromatography-mass spectrometry. J Chromatogr B Anal Technol Biomed Life Sci 2006; 834: 98–107.

124.

Wang

Pan

Lin

. [Simultaneous determination of clozapine, olanzapine and mirtazapine in human plasma by LC-MS/MS]. Fa Yi Xue Za Zhi 2009; 25: 123–126.

125.

Zhou

. Simultaneous determination of clozapine, olanzapine, risperidone and quetiapine in plasma by high-performance liquid chromatography-electrospray ionization mass spectrometry. J Chromatogr B Anal Technol Biomed Life Sci 2004; 802: 257–262.

126.

Awidi

Salem

Najib

. Determination of imatinib plasma levels in patients with chronic myeloid leukemia by high performance liquid chromatography-ultraviolet detection and liquid chromatography-tandem mass spectrometry: methods' comparison. Leuk Res 2010; 34: 714–717.

127.

Chahbouni

den Burger

Vos

. Simultaneous quantification of erlotinib, gefitinib, and imatinib in human plasma by liquid chromatography tandem mass spectrometry. Ther Drug Monit 2009; 31: 683–687.

128.

De Francia

D'Avolio

De Martino

. New HPLC-MS method for the simultaneous quantification of the antileukemia drugs imatinib, dasatinib, and nilotinib in human plasma. J Chromatogr B Anal Technol Biomed Life Sci 2009; 877: 1721–1726.

129.

Haouala

Zanolari

Rochat

. Therapeutic drug monitoring of the new targeted anticancer agents imatinib, nilotinib, dasatinib, sunitinib, sorafenib and lapatinib by LC tandem mass spectrometry. J Chromatogr B Anal Technol Biomed Life Sci 2009; 877: 1982–1996.

130.

Roche

McMahon

Clynes

. Development of a high-performance liquid chromatographic-mass spectrometric method for the determination of cellular levels of the tyrosine kinase inhibitors lapatinib and dasatinib. J Chromatogr B Anal Technol Biomed Life Sci 2009; 877: 3982–3990.

131.

Titier

Picard

Ducint

. Quantification of imatinib in human plasma by high-performance liquid chromatography-tandem mass spectrometry. Ther Drug Monit 2005; 27: 634–640.

132.

Bunch

Heideloff

Ritchie

. A fast and simple assay for busulfan in serum or plasma by liquid chromatography-tandem mass spectrometry using turbulent flow online extraction technology. J Chromatogr B Anal Technol Biomed Life Sci 2010; 878: 3255–3258.

133.

Moon

Lim

Hong

. Quantification of human plasma-busulfan concentration by liquid chromatography-tandem mass spectrometry. Ann Lab Med 2014; 34: 7–14.

134.

Choi

Bae

Park

. Simultaneous quantification of caffeine and its three primary metabolites in rat plasma by liquid chromatography-tandem mass spectrometry. Food Chem 2013; 141: 2735–2742.

135.

Rybak

Pao

Pfeiffer

. Determination of urine caffeine and its metabolites by use of high-performance liquid chromatography-tandem mass spectrometry: estimating dietary caffeine exposure and metabolic phenotyping in population studies. Anal Bioanal Chem 2014; 406: 771–784.

136.

Kuhn

Gotting

Kleesiek

. Simultaneous measurement of amiodarone and desethylamiodarone in human plasma and serum by stable isotope dilution liquid chromatography-tandem mass spectrometry assay. J Pharm Biomed Anal 2010; 51: 210–216.

137.

Lafuente-Lafuente

Alvarez

Leenhardt

. Amiodarone concentrations in plasma and fat tissue during chronic treatment and related toxicity. Br J Clin Pharmacol 2009; 67: 511–519.

138.

Bertol

Vaiano

Furlanetto

. Cross-reactivities and structure-reactivity relationships of six benzodiazepines to EMIT((R)) immunoassay. J Pharm Biomed Anal 2013; 84: 168–172.

139.

Fisichella

Morini

Sempio

. Validation of a multi-analyte LC-MS/MS method for screening and quantification of 87 psychoactive drugs and their metabolites in hair. Anal Bioanal Chem 2014; 406: 3497–3506.

140.

Maurer

. Multi-analyte procedures for screening for and quantification of drugs in blood, plasma, or serum by liquid chromatography-single stage or tandem mass spectrometry (LC-MS or LC-MS/MS) relevant to clinical and forensic toxicology. Clin Biochem 2005; 38: 310–318.

141.

Mueller

Weinmann

Dresen

. Development of a multi-target screening analysis for 301 drugs using a QTrap liquid chromatography/tandem mass spectrometry system and automated library searching. Rapid Commun Mass Spectrom 2005; 19: 1332–1338.

142.

Dresen

Gergov

Politi

. ESI-MS/MS library of 1,253 compounds for application in forensic and clinical toxicology. Anal Bioanal Chem 2009; 395: 2521–2526.

143.

Sauvage

Saint-Marcoux

Duretz

. Screening of drugs and toxic compounds with liquid chromatography-linear ion trap tandem mass spectrometry. Clin Chem 2006; 52: 1735–1742.

144.

Politi

Morini

Polettini

. A direct screening procedure for diuretics in human urine by liquid chromatography-tandem mass spectrometry with information dependent acquisition. Clin Chim Acta 2007; 386: 46–52.

145.

Al-Saffar

Stephanson

Beck

. Multicomponent LC-MS/MS screening method for detection of new psychoactive drugs, legal highs, in urine-experience from the Swedish population. J Chromatogr B Analyt Technol Biomed Life Sci 2013; 930: 112–120.

146.

Ojanpera

Pelander

Pelzing

. Isotopic pattern and accurate mass determination in urine drug screening by liquid chromatography/time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 2006; 20: 1161–1167.

147.

Broecker

Herre

Wust

. Development and practical application of a library of CID accurate mass spectra of more than 2,500 toxic compounds for systematic toxicological analysis by LC-QTOF-MS with data-dependent acquisition. Anal Bioanal Chem 2011; 400: 101–117.

148.

Borrey

Moerman

Cockx

. Column-switching LC-MS/MS analysis for quantitative determination of testosterone in human serum. Clin Chim Acta 2007; 382: 134–137.

149.

Søeborg

Frederiksen

Fruekilde

. Serum concentrations of DHEA, DHEAS, 17α-hydroxyprogesterone, Δ4-androstenedione and testosterone in children determined by TurboFlow-LC-MS/MS. Clin Chim Acta 2013; 419: 95–101.

150.

Buttler

Kruit

Blankenstein

. Measurement of dehydroepiandrosterone sulphate (DHEAS): a comparison of isotope-dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) and seven currently available immunoassays. Clin Chim Acta 2013; 424: 22–26.

151.

Farrell

Martin

McWhinney

. State-of-the-art vitamin D assays: a comparison of automated immunoassays with liquid chromatography-tandem mass spectrometry methods. Clin Chem 2012; 58: 531–542.

152.

Saenger

Laha

Bremner

. Quantification of serum 25-hydroxyvitamin D(2) and D(3) using HPLC-tandem mass spectrometry and examination of reference intervals for diagnosis of vitamin D deficiency. Am J Clin Pathol 2006; 125: 914–920.

153.

Shah

James

Barker

. Misleading measures in vitamin D analysis: a novel LC-MS/MS assay to account for epimers and isobars. Nutr J 2011; 10: 1–9.

154.

van den Ouweland

Beijers

Demacker

. Measurement of 25-OH-vitamin D in human serum using liquid chromatography tandem-mass spectrometry with comparison to radioimmunoassay and automated immunoassay. J Chromatogr B Anal Technol Biomed Life Sci 2010; 878: 1163–1168.

155.

Hosogi

Tanaka

Fujita

. LC-MS/MS coupled with immunoaffinity extraction for determination of estrone, 17beta-estradiol and estrone 3-sulfate in human plasma. J Chromatogr B Anal Technol Biomed Life Sci 2010; 878: 222–227.

156.

Kushnir

Rockwood

Bergquist

. High-sensitivity tandem mass spectrometry assay for serum estrone and estradiol. Am J Clin Pathol 2008; 129: 530–539.

157.

. Simultaneous determination of norethindrone and ethinyl estradiol in human plasma by high performance liquid chromatography with tandem mass spectrometry–experiences on developing a highly selective method using derivatization reagent for enhancing sensitivity. J Chromatogr B Anal Technol Biomed Life Sci 2005; 825: 223–232.

158.

Nelson

Grebe

OKane

. Liquid chromatography-tandem mass spectrometry assay for simultaneous measurement of estradiol and estrone in human plasma. Clin Chem 2004; 50: 373–384.

159.

Owen

Keevil

. A rapid direct assay for the routine measurement of oestradiol and oestrone by liquid chromatography tandem mass spectrometry. Ann Clin Biochem 2014; 51: 360–367.

160.

Etter

Eichhorst

Lehotay

. Clinical determination of 17-hydroxyprogesterone in serum by LC-MS/MS: comparison to Coat-A-Count RIA method. J Chromatogr B Anal Technol Biomed Life Sci 2006; 840: 69–74.

161.

Huang

Kalhorn

Baillie

. Determination of free and total cortisol in plasma and urine by liquid chromatography-tandem mass spectrometry. Ther Drug Monit 2007; 29: 215–224.

162.

Monaghan

Owen

Trainer

. Comparison of serum cortisol measurement by immunoassay and liquid chromatography-tandem mass spectrometry in patients receiving the 11β-hydroxylase inhibitor metyrapone. Ann Clin Biochem 2011; 48: 441–446.

163.

McCann

Gillingwater

Keevil

. Measurement of urinary free cortisol using liquid chromatography-tandem mass spectrometry: comparison with the urine adapted ACS:180 serum cortisol chemiluminescent immunoassay and development of a new reference range. Ann Clin Biochem 2005; 42: 112–118.

164.

Taylor

Machacek

Singh

. Validation of a high-throughput liquid chromatography-tandem mass spectrometry method for urinary cortisol and cortisone. Clin Chem 2002; 48: 1511–1519.

165.

Turpeinen

Stenman

. Determination of urinary free cortisol by liquid chromatography-tandem mass spectrometry. Scand J Clin Lab Invest 2003; 63: 143–150.

166.

Wear

Owen

Duxbury

. A simplified method for the measurement of urinary free cortisol using LC-MS/MS. J Chromatogr B Anal Technol Biomed Life Sci 2007; 858: 27–31.

167.

Middle

. Dehydroepiandrostenedione sulphate interferes in many direct immunoassays for testosterone. Ann Clin Biochem 2007; 44: 173–177.

168.

French

. Development and validation of a serum total testosterone liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay calibrated to NIST SRM 971. Clin Chim Acta 2013; 415: 109–117.

169.

Methlie

Hustad

Kellmann

. Multisteroid LC-MS/MS assay for glucocorticoids and androgens, and its application in Addison's disease. Endocr Connect 2013; 2: 125–136.

170.

Handelsman

Wartofsky

. Requirement for mass spectrometry sex steroid assays in the Journal of Clinical Endocrinology and Metabolism. J Clin Endocrinol Metab 2013; 98: 3971–3973.

171.

Editorial Board J. Letter of concern. J Clin Endocrinol Metab 2014; 99: 1–1.

172.

Fanelli

Belluomo

Di Lallo

. Serum steroid profiling by isotopic dilution-liquid chromatography-mass spectrometry: comparison with current immunoassays and reference intervals in healthy adults. Steroids 2011; 76: 244–253.

173.

Jung

Torpy

. A longitudinal study of plasma and urinary cortisol in pregnancy and postpartum. J Clin Endocrinol Metab 2011; 96: 1533–1540.

174.

Aso

Guerrero

Cekan

. A rapid 5 hour radioimmunoassay of progesterone and oestradiol in human plasma. Clin Endocrinol 1975; 4: 173–182.

175.

Barnard

Hennam

Collins

. Further studies on radioimmunoassay systems for plasma oestradiol. J Steroid Biochem 1975; 6: 107–116.

176.

Bayard

Beitins

Kowarski

. Measurement of plasma aldosterone by radioimmunoassay. J Clin Endocrinol Metab 1970; 31: 1–6.

177.

Mayes

Furuyama

Kem

. A radioimmunoassay for plasma aldosterone. J Clin Endocrinol Metab 1970; 30: 682–685.

178.

Gabler

Miller

Wang

. A simple liquid chromatography-tandem mass spectrometry method for measuring metanephrine and normetanephrine in urine. Clin Chem Lab Med 2011; 49: 1213–1216.

179.

Peitzsch

Pelzel

Glockner

. Simultaneous liquid chromatography tandem mass spectrometric determination of urinary free metanephrines and catecholamines, with comparisons of free and deconjugated metabolites. Clin Chim Acta 2013; 418: 50–58.

180.

Taylor

Singh

. Validation of liquid chromatography-tandem mass spectrometry method for analysis of urinary conjugated metanephrine and normetanephrine for screening of pheochromocytoma. Clin Chem 2002; 48: 533–539.

181.

Lagerstedt

O'Kane

Singh

. Measurement of plasma free metanephrine and normetanephrine by liquid chromatography-tandem mass spectrometry for diagnosis of pheochromocytoma. Clin Chem 2004; 50: 603–611.

182.

Marney

Laha

Baird

. Isopropanol protein precipitation for the analysis of plasma free metanephrines by liquid chromatography-tandem mass spectrometry. Clin Chem 2008; 54: 1729–1732.

183.

Peaston

Graham

Chambers

. Performance of plasma free metanephrines measured by liquid chromatography-tandem mass spectrometry in the diagnosis of pheochromocytoma. Clin Chim Acta 2010; 411: 546–552.

184.

Peitzsch

Prejbisz

Kroiss

. Analysis of plasma 3-methoxytyramine, normetanephrine and metanephrine by ultraperformance liquid chromatography-tandem mass spectrometry: utility for diagnosis of dopamine-producing metastatic phaeochromocytoma. Ann Clin Biochem 2013; 50: 147–155.

185.

Petteys

Graham

Parnas

. Performance characteristics of an LC-MS/MS method for the determination of plasma metanephrines. Clin Chim Acta 2012; 413: 1459–1465.

186.

Eisenhofer

Lenders

Siegert

. Plasma methoxytyramine: a novel biomarker of metastatic pheochromocytoma and paraganglioma in relation to established risk factors of tumour size, location and SDHB mutation status. Eur J Cancer 2012; 48: 1739–1749.

187.

Bystrom

Salameh

Reitz

. Plasma renin activity by LC-MS/MS: development of a prototypical clinical assay reveals a subpopulation of human plasma samples with substantial peptidase activity. Clin Chem 2010; 56: 1561–1569.

188.

Chappell

McAvoy

Weiss

. Development and validation of an ultra-sensitive method for the measurement of plasma renin activity in human plasma via LC-MS/MS. Bioanalysis 2012; 4: 2843–2850.

189.

Chace

DiPerna

Mitchell

. Electrospray tandem mass spectrometry for analysis of acylcarnitines in dried postmortem blood specimens collected at autopsy from infants with unexplained cause of death. Clin Chem 2001; 47: 1166–1182.

190.

Chace

Hillman

Van Hove

. Rapid diagnosis of MCAD deficiency: quantitative analysis of octanoylcarnitine and other acylcarnitines in newborn blood spots by tandem mass spectrometry. Clin Chem 1997; 43: 2106–2113.

191.

Research NNIfH. Expanded newborn screening: an information resource for parents and professionals on the expanded newborn screening program and rare conditions, http://www.expandedscreening.org/site/home/start.asp (2014, accessed 21 October 2014).

192.

Dhillon

Bhandal

Aznar

. Improved tandem mass spectrometry (MS/MS) derivatized method for the detection of tyrosinemia type I, amino acids and acylcarnitine disorders using a single extraction process. Clin Chim Acta 2011; 412: 873–879.

193.

Naylor

Chace

. Automated tandem mass spectrometry for mass newborn screening for disorders in fatty acid, organic acid, and amino acid metabolism. J Child Neurol 1999; 14(Suppl 1): S4–S8.

194.

Jeong

Kim

Park

. Amino acid analysis of dried blood spots for diagnosis of phenylketonuria using capillary electrophoresis-mass spectrometry equipped with a sheathless electrospray ionization interface. Anal Bioanal Chem 2013; 405: 8063–8072.

195.

la Marca

Malvagia

Materazzi

. LC-MS/MS method for simultaneous determination on a dried blood spot of multiple analytes relevant for treatment monitoring in patients with tyrosinemia type I. Anal Chem 2012; 84: 1184–1188.

196.

Haas

Gan-Schreier

Langhans

. Differential diagnosis in patients with suspected bile acid synthesis defects. World J Gastroenterol 2012; 18: 1067–1076.

197.

Stanislaus

Guo

. Development of an isotope labeling ultra-high performance liquid chromatography mass spectrometric method for quantification of acylglycines in human urine. Anal Chim Acta 2012; 750: 161–172.

198.

Rauh

. Steroid measurement with LC-MS/MS. Application examples in pediatrics. J Steroid Biochem Mol Biol 2010; 121: 520–527.

199.

Seo

Park

Kim

. Steroid profiling for congenital adrenal hyperplasia by tandem mass spectrometry as a second-tier test reduces follow-up burdens in a tertiary care hospital: a retrospective and prospective evaluation. J Perinatal Med 2014; 42: 121–127.

200.

McWhinney

Briscoe

Ungerer

. Measurement of cortisol, cortisone, prednisolone, dexamethasone and 11-deoxycortisol with ultra high performance liquid chromatography-tandem mass spectrometry: application for plasma, plasma ultrafiltrate, urine and saliva in a routine laboratory. J Chromatogr B Anal Technol Biomed Life Sci 2010; 878: 2863–2869.

201.

Couchman

Belsey

Handley

. A novel approach to quantitative LC-MS/MS: therapeutic drug monitoring of clozapine and norclozapine using isotopic internal calibration. Anal Bioanal Chem 2013; 405: 9455–9466.

202.

Robandt

Reda

Klette

. Complete automation of solid-phase extraction with subsequent liquid chromatography-tandem mass spectrometry for the quantification of benzoylecgonine, m-hydroxybenzoylecgonine, p-hydroxybenzoylecgonine, and norbenzoylecgonine in urine–application to a high-throughput urine analysis laboratory. J Anal Toxicol 2008; 32: 577–585.

203.

Tellez

Mamikunian

O'Dorisio

. A single fasting plasma 5-HIAA value correlates with 24-hour urinary 5-HIAA values and other biomarkers in midgut neuroendocrine tumors (NETs). Pancreas 2013; 42: 405–410.

204.

Chadwick

Keevil

. Measurement of cotinine in urine by liquid chromatography tandem mass spectrometry. Ann Clin Biochem 2007; 44: 455–462.

205.

Heavner

Richardson

Morgan

. Validation and application of a method for the determination of nicotine and five major metabolites in smokers' urine by solid-phase extraction and liquid chromatography-tandem mass spectrometry. Biomed Chromatogr 2005; 19: 312–328.

206.

Piller

Gilch

Scherer

. Simple, fast and sensitive LC-MS/MS analysis for the simultaneous quantification of nicotine and 10 of its major metabolites. J Chromatogr B Anal Technol Biomed Life Sci 2014; 951–952: 7–15.

207.

Shu

Wang

. Simultaneous serum nicotine, cotinine, and trans-3'-hydroxycotinine quantitation with minimal sample volume for tobacco exposure status of solid organ transplant patients. J Chromatogr B Anal Technol Biomed Life Sci 2013; 928: 139–145.

208.

Yuan

Kosewick

Wang

. A simple, fast, and sensitive method for the measurement of serum nicotine, cotinine, and nornicotine by LC-MS/MS. J Sep Sci 2013; 36: 2394–2400.

209.

Chen

Qin

. Quantitative determination of azithromycin in human plasma by ultra performance liquid chromatography-electrospray ionization mass spectrometry and its application in a pharmacokinetic study. J Chromatogr B Anal Technol Biomed Life Sci 2007; 855: 255–261.

210.

Mutters

Hodiamont

de Jong

. Performance of Kiestra total laboratory automation combined with MS in clinical microbiology practice. Ann Lab Med 2014; 34: 111–117.

211.

Peng

Yang

Xiong

. Sensitive and rapid detection of viruses associated with hand foot and mouth disease using multiplexed MALDI-TOF analysis. J Clin Virol 2013; 56: 170–174.

212.

Alatoom

Cunningham

Ihde

. Comparison of direct colony method versus extraction method for identification of gram-positive cocci by use of Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol 2011; 49: 2868–2873.

213.

Saffert

Cunningham

Ihde

. Comparison of Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometer to BD Phoenix automated microbiology system for identification of gram-negative bacilli. J Clin Microbiol 2011; 49: 887–892.

214.

Honour

. Development and validation of a quantitative assay based on tandem mass spectrometry. Ann Clin Biochem 2011; 48: 97–111.

215.

Khatami Z, Hill R, Sturgeon C, et al. Measurement verification in the clinical laboratory, http://www.acb.org.uk/docs/default-source/committees/scientific/guidelines/measurement-verification/Measurement_verification_final_090608.pdf?sfvrsn=0 (2009, accessed 20 October 2014).

216.

Joint Commission on Accreditation of Healthcare organization. ACCEPTED: new and revised laboratory requirements to meet relevant CLIA regulations. Jt Comm Perspect 2012; 32: 3–3.

217.

Blume

Brendel

Brudny-Kloppel

. Workshop/conference report on EMA draft guideline on validation of bioanalytical methods. Eur J Pharm Sci 2011; 42: 300–305.

218.

Smith

. Bioanalytical method validation: notable points in the 2009 draft EMA guideline and differences with the 2001 FDA guidance. Bioanalysis 2010; 2: 929–935.

219.

US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research and Center for Veterinary medicine. Guidance for industry: bioanalytical method validation, http://www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf (2001, accessed 20 October 2014).

220.

Rauh

. LC-MS/MS for protein and peptide quantification in clinical chemistry. J Chromatogr B Anal Technol Biomed Life Sci 2012; 883–884: 59–67.

221.

de Jong

de Vries

Kema

. Current status and future developments of LC-MS/MS in clinical chemistry for quantification of biogenic amines. Clin Biochem 2011; 44: 95–103.

222.

Halladay

Delarosa

Tran

. High-throughput, 384-well, LC-MS/MS CYP inhibition assay using automation, cassette-analysis technique, and streamlined data analysis. Drug Metab Lett 2011; 5: 220–230.

223.

Watt

Morrison

Locker

. Higher throughput bioanalysis by automation of a protein precipitation assay using a 96-well format with detection by LC-MS/MS. Anal Chem 2000; 72: 979–984.

224.

Zhang

Wei

Ayres

. An automation-assisted generic approach for biological sample preparation and LC-MS/MS method validation. Bioanalysis 2011; 3: 1975–1986.

225.

Wohleb RGR, Geyer R and Eckard S. Technical background to the Tecan AC extraction plate, http://www.tecan.com/platform/apps/checkdoc/index.asp?doc_Id=2123,538&prot=1,1 (2013, accessed 20 October 2014).

226.

Konig

Goethel

Rusu

. Deproteination of serum samples for LC-MS/MS analyses by applying magnetic micro-particles. Clin Biochem 2013; 46: 652–655.

227.

Konig

Vogeser

. Sample preparation for measurement of plasma mycophenolic acid concentrations using chromatographically functionalized magnetic micro-particles. Eur J Mass Spectrom (Chichester, Eng) 2012; 18: 413–417.

228.

Roberts PH. Online automated protein crash, solid phase extraction and anaysis of Vitamin D in serum using Instrument Top Sample Prep (ITSP), Gerstel Multi Purpose Sample and Agilent LC-MS/MS. Anatune Chromatography Technical Note No AS97, http://www.friiscan.co.uk/sites/default/files/AS97.PDF (2010, accessed 15 October 2014).

229.

Roberts PH. Automating the analysis of selected immunosuppressants in whole blood using a GERSTEL multi purpose sampler coupled to liquid chromatography tandem mass spectrometry detection. Anatune Chromatography Technical Note No AS102, http://www.friiscan.co.uk/sites/default/files/AS102.pdf (2011, accessed 15 October 2014).

230.

Roberts PGB. Automating the analysis of androstenedione and testosterone in serum using a GERSTEL multi purpose sampler coupled to liquid chromatography tandem mass spectrometry detection. Anatune Chromatography Technical Note No AS104, http://www.friiscan.co.uk/sites/default/files/AS104.pdf (2011, accessed 15 October 2014).

231.

Chaurand

Luetzenkirchen

Spengler

. Peptide and protein identification by matrix-assisted laser desorption ionization (MALDI) and MALDI-post-source decay time-of-flight mass spectrometry. J Am Soc Mass Spectrom 1999; 10: 91–103.

232.

Gevaert

De Mol

Puype

. Sample preparation procedures for ultrasensitive protein identification by PSD-MALDI-TOF mass spectrometry. J Protein Chem 1998; 17: 560–560.

233.

Jungblut

Thiede

. Protein identification from 2-DE gels by MALDI mass spectrometry. Mass Spectrom Rev 1997; 16: 145–162.

234.

Barnidge

Hall

Stocker

. Evaluation of a cleavable stable isotope labeled synthetic peptide for absolute protein quantification using LC-MS/MS. J Proteome Res 2004; 3: 658–661.

235.

Ewles

Turpin

Goodwin

. Validation of a bioanalytical method for the quantification of a therapeutic peptide, ramoplanin, in human dried blood spots using LC-MS/MS. Biomed Chromatogr 2011; 25: 995–1002.

236.

Nemirovskiy

Fountain

. Clinical validation of an immunoaffinity LC-MS/MS assay for the quantification of a collagen type II neoepitope peptide: a biomarker of matrix metalloproteinase activity and osteoarthritis in human urine. Anal Biochem 2007; 369: 41–53.

237.

Nemirovskiy

Szekely-Klepser

. Design and validation of an immunoaffinity LC-MS/MS assay for the quantification of a collagen type II neoepitope peptide in human urine: application as a biomarker of osteoarthritis. Methods Mol Biol 2010; 641: 253–270.

238.

Chambers

Legido-Quigley

Smith

. Development of a fast method for direct analysis of intact synthetic insulins in human plasma: the large peptide challenge. Bioanalysis 2013; 5: 65–81.

239.

Clarke

Zhang

Reitz

. A novel mass spectrometry-based assay for the accurate measurement of thyroglobulin from patient samples containing antithyroglobulin autoantibodies. J Investig Med 2012; 60: 1157–1163.

240.

Kushnir

Rockwood

Roberts

. Measurement of thyroglobulin by liquid chromatography-tandem mass spectrometry in serum and plasma in the presence of antithyroglobulin autoantibodies. Clin Chem 2013; 59: 982–990.

241.

Thomas

Kohler

Schanzer

. Determination of IGF-1 and IGF-2, their degradation products and synthetic analogues in urine by LC-MS/MS. Analyst 2011; 136: 1003–1012.

242.

Niederkofler

Phillips

Krastins

. Targeted selected reaction monitoring mass spectrometric immunoassay for insulin-like growth factor 1. PloS One 2013; 8: 25–30.

243.

Wilffert

Reis

Hermans

. Antibody-free LC-MS/MS quantification of rhTRAIL in human and mouse serum. Anal Chem 2013; 85: 10754–10760.

244.

Kirsch

Widart

Louette

. Development of an absolute quantification method targeting growth hormone biomarkers using liquid chromatography coupled to isotope dilution mass spectrometry. J Chromatogr A 2007; 1153: 300–306.

245.

Barnidge

Goodmanson

Klee

. Absolute quantification of the model biomarker prostate-specific antigen in serum by LC-Ms/MS using protein cleavage and isotope dilution mass spectrometry. J Proteome Res 2004; 3: 644–652.

246.

Hoofnagle

Becker

Wener

. Quantification of thyroglobulin, a low-abundance serum protein, by immunoaffinity peptide enrichment and tandem mass spectrometry. Clin Chem 2008; 54: 1796–1804.

247.

Arsene

Henrion

Diekmann

. Quantification of growth hormone in serum by isotope dilution mass spectrometry. Anal Biochem 2010; 401: 228–235.

248.

Krastins

Prakash

Sarracino

. Rapid development of sensitive, high-throughput, quantitative and highly selective mass spectrometric targeted immunoassays for clinically important proteins in human plasma and serum. Clin Biochem 2013; 46: 399–410.

249.

Lopez

Rezai

Sarracino

. Selected reaction monitoring-mass spectrometric immunoassay responsive to parathyroid hormone and related variants. Clin Chem 2010; 56: 281–290.

250.

Prakash

Rezai

Krastins

. Interlaboratory reproducibility of selective reaction monitoring assays using multiple upfront analyte enrichment strategies. J Proteome Res 2012; 11: 3986–3995.

251.

Schmidt

. Metabolomics takes its place as latest up-and-coming “omic” science. J Natl Cancer Inst 2004; 96: 732–734.

252.

Lin

Liu

Yang

. GC/MS-based metabolomics reveals fatty acid biosynthesis and cholesterol metabolism in cell lines infected with influenza A virus. Talanta 2010; 83: 262–268.

253.

Styczynski

Moxley

Tong

. Systematic identification of conserved metabolites in GC/MS data for metabolomics and biomarker discovery. Anal Chem 2007; 79: 966–973.

254.

Ceglarek

Leichtle

Brugel

. Challenges and developments in tandem mass spectrometry based clinical metabolomics. Mol Cell Endocrinol 2009; 301: 266–271.

255.

Lanza

Zhang

Ward

. Quantitative metabolomics by H-NMR and LC-MS/MS confirms altered metabolic pathways in diabetes. PloS One 2010; 5: 1–10.

256.

Stoop

Coulier

Rosenling

. Quantitative proteomics and metabolomics analysis of normal human cerebrospinal fluid samples. Mol Cell Proteom 2010; 9: 2063–2075.

257.

Yang

Sadilek

Lidstrom

. Streamlined pentafluorophenylpropyl column liquid chromatography-tandem quadrupole mass spectrometry and global (13)C-labeled internal standards improve performance for quantitative metabolomics in bacteria. J Chromatogr A 2010; 1217: 7401–7410.

258.

Jones

Last

. Rapid LC-MS/MS profiling of protein amino acids and metabolically related compounds for large-scale assessment of metabolic phenotypes. Methods Mol Biol 2012; 828: 1–11.

259.

Balog

Sasi-Szabo

Kinross

. Intraoperative tissue identification using rapid evaporative ionization mass spectrometry. Sci Transl Med 2013; 5: 1–11.

260.

Balog

Szaniszlo

Schaefer

. Identification of biological tissues by rapid evaporative ionization mass spectrometry. Anal Chem 2010; 82: 7343–7350.

261.

Sachfer

Szaniszlo

Gunther

. In situ, real-time identification of biological tissues by ultraviolet and infrared laser desorption ionization mass spectrometry. Anal Chem 2011; 83: 1632–1640.

262.

Schafer

Denes

Albrecht

. In vivo, in situ tissue analysis using rapid evaporative ionization mass spectrometry. Angewandte Chemie (International ed in English) 2009; 48: 8240–8242.

Liquid chromatography tandem mass spectrometry in the clinical laboratory

Abstract

Keywords

Introduction

Elements of LC-MS/MS operation

Ion production

Fragmentation

Transitions and scans

Sample preparation

Kits and calibration

Column choice and ion suppression

Standardization and reference method services

Internal standard choice

Matrices

Current clinical uses of MS

Therapeutic drug monitoring

Toxicology

Endocrinology

Paediatrics

Small molecule quantitation

Microbiology

Current analytical considerations

Validation

Batch versus random access

Emerging and future applications

Automated sample handling

Pay-per-sample

Peptide and protein quantitation

Metabolomics

Point of care testing

Total laboratory automation and random access

Conclusion

Footnotes

Acknowledgements

Declaration of conflicting interests

Funding

Ethical approval

Guarantor

Contributorship

References