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Abstract

Background:

Methamphetamine (MA) and phentermine (PTM) are critical isomeric compounds that often coexist in biological specimen in toxicology cases which have similar nominal mass. The dependency of high-resolution mass spectrometer and special chiral stationary phase column for separation of isomeric compound impedes screening throughput especially in meeting high sampling demand.

Aim:

In the absence of such equipment for identification, we introduce the flow injection analysis (FIA)-MRM³ technique and demonstrated its capabilities for quick identification of both isomeric compounds without liquid chromatography (LC) separation through the addition of selectivity criteria in the MRM³ mode.

Method:

20 to 100 ng/mL of MA and PTM, respectively, in whole blood sample (WBS) and dried blood stain (DBS) were used to develop the method for the identification and relative quantification. Twenty whole blood samples which were reported positive of the drugs were randomly selected and were individually stained onto the Flinders Technology Associates (FTA) card for DBS study.

Results:

The peaks from the two isomeric compounds were successfully discerned for all the tested specimens using the FIA-MRM³ spectrometry technique. The limit of detection (LOD) and limit of quantification (LOQ) for MA and PTM were comparable to normal liquid chromatography-tandem mass spectrometry runs at 2.23 and 2.07 ng/mL, respectively, for WBS (n = 30). For DBS, the LOD and LOQ were 3.40 and 2.86 ng/mL, respectively. The accuracy and inter-day precision for DBS and WBS were in the range of 99.97–111.19% and 7.17–9.55%, respectively.

Conclusion:

These results demonstrated that the technique is highly adoptable in the screening of isomeric compounds of similar masses in a simple and rapid analysis.

Keywords

FIA (flow injection analysis)high-throughput screening isomeric compounds methamphetamine phentermine WBS DBS

Introduction

Street methamphetamine (MA) is an illicit substance, while phentermine (PTM) is a weight management drug, and misinterpretation of MA versus PTM can result in serious implications. Both MA and PTM are isomeric compounds with similar molecular formula but different chemical structures. Isomeric compounds give similar parent and fragment ions mass-to-charge ratio (m/z) and gas-phase ion chemistry, resulting in identical m/z values for the precursor and fragment ions. Prime example of critical isomeric amphetamine-type stimulants are MA and PTM, which often coexist in biological specimen in toxicology cases. The summary of physical properties and chemical structures for both compounds is shown in Table 1.

Table 1.

Physical properties and chemical structures of MA and PTM.

Compound	Molecular formula	Chemical structure	Monoisotopic mass (g/mol)
MA	C₁₀H₁₅N		149.12
PTM	C₁₀H₁₅N		149.12

MA: methamphetamine; PTM: phentermine.

In Malaysia, MA is listed under both Poison Act (PA) 1952 and Dangerous Drugs Act (DDA) 1952. While PTM is a prescription medicine for treating obesity^1,2 in weight management programme and listed under the PA 1952 as a controlled substance. Substances listed under DDA 1952 carries heavier penalties chargeable under Criminal Procedure Code 399 in the legislation for forensic and medicolegal cases. In Royal Malaysia Armed Forces, MA and other drugs abusers can be charged under Armed Forces Act 1972. Liquid (LC) or gas chromatography (GC) are the gold standard for the identification and quantification of drugs of abuse and metabolites in biological specimens.³ Elaborate sample preparations and lengthy analysis time are the two main challenges often faced by toxicology analyst/prosecutor in meeting with the demand of high-throughput analysis.^4
–6

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly being used in clinical and forensic toxicology for the determination and quantification of a wide range of compounds, including differentiation of isomeric compounds in biological samples.⁷ Variation within these methods focused primarily on LC conditions such as the type of column and selection of mobile phase solvent system. The effect of modifier was also reported to increase LC separation and gave better peak shape in quantitative analysis.⁸

Flow injection analysis (FIA) in combination with tandem mass spectrometry (MS) is a well-known technique firmly established in many biomedical laboratories.⁹ FIA, also termed loop injection, is an alternative approach to LC-MS/MS in which the analytical column is removed and detection or separation occurs within the MS instrument. The method simply utilizes a small amount of sample, introduced into a stream of mobile phase solution which is then delivered continuously into the mass spectrometer. In such cases, the analyte response rather than chromatographic separation is used for quantification. In typical LC-MS/MS methods, separation of isomeric compounds requires longer run time with specilized column (stationary phase) and high-resolution MS instrumentation.^2,10,11

The FIA-MS methods were successfully applied in many cases across the sciences including the work of Johnson,¹² Sander et al.,¹³ and Niesser et al.,¹⁴ who have independantly developed FIA-MS techniques for the analysis of endogenous compounds in human body fluids for medical diagnostics and clinical applications. The capability of this technique has been adopted in the applications of forensic toxicology, for example,determination of cocaine and opiates in human hair samples,¹⁵ organophosphorus pesticides in porcine¹⁶ as well as in the pharmacology such as determination of metformin in dog serum after intravenous injection in which analysis time is remarkably shorten to 2 min.¹⁷ In addition, a FIA-MS/MS method was reported as comparable to enzymatic assay for creatinine determination in human.¹⁸ In this study, we have developed and demonstrated an alternative fast screening technique on top of confirmatory chromatography screening for isomeric compounds, that is, MA and PTM using FIA-MRM³ spectrometry. The selectivity of MRM³ towards targeted transitions provided additional identification dimension to substitute the absence of retention time information. The developed FIA-MRM³ method offers high-throughput screening prior to routine chromatographic-MS analysis reducing overall analysis time up to 80%, resulting in significant reduction of solvent consumptions and enhanced column lifetime without compromising on sensitivity and selectivity. The developed and validated method was successfully applied in whole blood and dried blood stain (DBS) medium for forensic toxicology cases in Malaysia.

Materials and methods

Generals

Standards: MA, PTM and methamphetamine-d₁₄ (MA-d₁₄) were purchased from Lipomed (Switzerland). Acetonitrile and methanol were HPLC grade (Merck, Darmstadt, Germany). Chlorobutane was acquired from Fischer Chemical (Loughborough, Leicestershire, UK). Formic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA). Whatman^® FTA^® card was purchased from Merck (Darmstadt, Germany). Ultrapure water was from arium^® pro UV Ultrapure Water with a specific resistance at > 18 MΩcm.

Sample collection and pretreatment

Blank blood samples were obtained from bull’s blood. The blood was tested earlier and used in the preparation of calibration solutions and matrix matched analysis. Calibration solutions for MA and PTM were prepared in blank blood at six different concentrations of 5, 10, 20, 50, 100 and 200 ng/mL. Seven (7) replicates with concentration of 20 and 100 ng/mL, respectively, were prepared in WBS and DBS for method validation. Phosphate buffer (20 mM, pH 7.4) was prepared by dissolving monobasic sodium phosphate in water, followed by adding sodium hydroxide to adjust the pH (phosphate buffer releases Hb from the stain and lead to best recovery).

Whole blood samples (n = 20) with positive identification of MA/PTM were obtained from various real cases submitted for forensic and medicolegal analysis in the period from January 2016 until September 2017 to Forensic Division, Department of Chemistry Malaysia. The whole blood samples (n = 20) were individually stained onto Whatman FTA for DBS analysis.

External quality controls (QCs) consisted of whole blood samples tested for Proficiency Testing (PT) provided by College of American Pathologists (CAP), USA. Whatman FTA card was selected as DBS medium and all samples were stored at 4°C until the time of analysis.

FIA-MRM³ spectrometry conditions

FIA-MRM³ spectrometry was performed using an Exion LC SCIEX Binary SL Series System interfaced to SCIEX 5500 Q TRAP system (Toronto, Canada) equipped with a TurboIonspray^TM interface. MDS Sciex Analyst Software (Version 1.6.3) together with MultiQuant Software (Version 3.0.2) were used during method development, data acquisition, data processing and statistical analysis.

A sample volume of 2 μL was injected using an Exion LC SCIEX autoinjector set to 10°C with a sampling speed of 5.0 μL/s and was delivered through a union connector with an isocratic mobile phase consisted of water/acetonitrile (50:50, v/v) with 0.1% formic acid at a flow rate of 0.2 mL/min for a run time of 2 min. MRM³ was achieved by using electrospray ionization (ESI) source in the positive ion mode. The method was performed via MRM-Information Dependant Acquisition (IDA)-MRM³.

Optimization on the first and second MRM transitions, corresponding to the confirmation and quantification, were conducted via manual infusion to achieve maximum sensitivity. The parameters optimized for the MRM transitions were declustering potential (DP), entrance potential (EP), collision energy (CE) and collision exit potential (CXP). Similarly, on the MRM³ experiment, the product ions were optimized using the second precursor ions of the MRM confirmation transition. Using the optimized compound settings from the MS/MS, the intensity for the product mass spectra from the second precursors were monitored and recorded.

The excitation energy (AF2) was ramped from 10 to 200 mV to achieve the highest product signal counts. The AF2 parameters equates to the fragmentation voltage energy transferred towards the isolated second precursor ions in the linear ion trap (LIT). The MS/MS/MS (MS³) spectra were also collected during MRM³ optimization and updated into the software database package. The monitored ion transitions in MRM³ for the tested analytes were 150/119/91 Da (MA), 150/133/91 Da (PTM) and 164/130 Da (ISTD MA-d₁₄). Optimization for sensitivity was also conducted on the source settings and the optimization parameters were summarized in Table 2.

Table 2.

Optimization of source and parameters on MRM-IDA-MRM³ settings.

		MA	PTM
Source settings	Heater temperature (°C)	550
	Ion spray voltage (V)	5500
	Nebulizing gas (Psi)	40
	Heater gas pressure (Psi)	40
	Collision gas pressure	Low
Compound settings	DP (V)	60	56
	EP (V)	10	10
	CE (V)	14^a, 23^b	12^c, 23^d
	CXP (V)	9	9
MRM³ settings	AF2 (mV)	85	62

DP: declustering potential; EP: entrance potential; CE: collision potential; CXP: collision exit potential; AF2: excitation energy; MA: methamphetamine; PTM: phentermine; IDA: information dependant acquisition.

MRM transition corresponding to: ^a150 > 119; ^b150 > 91; ^c150 > 133; ^d150 > 91.

Whole blood sample (WBS) preparation

Whole blood samples (WBS) were subjected to liquid–liquid extraction (LLE). An aliquot (30 μL) of the internal standard; MA-d₁₄ solution was added into 1 mL of the samples, followed by 0.5 mL of phosphate buffer solution and 3 mL of 1-chlorobutane. The samples were then equilibrated on a roller mixer for approximately 1 h followed by centrifugation at 2000 r/min for 10 min. The upper organic solvent layer was transferred to a clean tube and dried using rotary evaporator. The dried extract was reconstituted in 100 μL of mobile phase solution and transferred to an autosampler vial for FIA-MRM³ spectrometry analysis.

DBS sample preparation

Blood stains were prepared by spotting 100 μL aliquot of blood onto Whatman FTA card, which were subsequently dried at room temperature overnight. The stained FTA cards were put into test tubes followed by adding 30 μL of internal standard; MA-d₁₄ solution. LLE was performed by adding 1 mL of water followed by 0.5 mL phosphate buffer and 3 mL of 1-chlorobutane. The subsequent steps were similar to that of whole blood samples preparation.

Method validation

Method validation was performed as outlined by the Scientific Working Group for Forensic Toxicology (SWGTOX) Standard Practices for Method Validation in Forensic Toxicology¹⁹ and UNODC Guidance for the Validation of Analytical Methodology and Calibration of Equipment used for Testing of Illicit Drugs in Seized Materials and Biological Samples.²⁰ Parameters evaluated in method validation include linearity, specificity/selectivity and matrix effect, LOD and LOQ, accuracy and precision (within the laboratory repeatability and/or within the laboratory reproducibility conditions) to determine robustness and carry over.

The linearity was tested using a standard solution of MA and PTM with the concentration from 5 to 200 ng/mL in-line with the level of tested analytes (normal MA level: 0.01–0.05 μg/mL and therapeutic PTM level: 0.09–0.51 μg/mL).²¹ Calibration curves were constructed using the peak height ratio of the analyte and internal standard, plotted against the corresponding concentration using linear regression with an 1/x weighting factor. The reagent-only calibration standards and matrix-matched calibration standards were used to assess the matrix effects.

The LOD and LOQ were performed to establish method sensitivity. LOD is defined as the lowest concentration of the analyte that resulted in signal-to-noise (S/N) ratio of 3:1 while the LOQ is defined as the lowest concentration of the analyte that resulted in S/N ratio of 10:1. Specificity of the proposed method was assessed by analysing the response in both blank and control samples.

The accuracy of the method was expressed as average recoveries of spiked blank matrix at 20 and 100 ng/mL concentration levels. Precision of the method was presented as relative standard deviation (RSD%) of within-laboratory reproducibility analyses. Seven replicates for each set were analysed during each working day to test the intra-day precision and subsequently during three consecutive days for inter-day precision. Robustness was assessed by making deliberate variations to the method (duration of manual shaking, vortex shaking and centrifugation), and the subsequent effects on method performance (accuracy, precision) were investigated.

In order to assess the carry over parameter, blank samples were analysed immediately after a high concentration of spiked standard of drugs. For this evaluation, blank sample with Internal standards (ISTD) and blank solvents were analysed after injection of 200 ng/mL calibrator and after each of five replicates injection of 100 ng/mL in a batch run.

Application to real samples

The developed method was applied to authentic case samples (n = 20) involving whole blood samples stained onto Whatman FTA card to make DBS medium. A random selection of drug-positive casework for MA/PTM parameters that had already been characterized by conventional means involving Enzyme-Linked Immunosorbent Assay (ELISA), GC-MS and LC-MS/MS (MRM mode) was chosen for FIA-MRM³ analysis. The external QCs from PT provided by College of American Pathologists (CAP), USA that had been accredited under ISO 17025 and International American Society of Crime Lab Directors – Laboratories Accreditation Board (ASCLD-LAB) accreditations were also tested to prove the reliability of the method for future use in forensic toxicology field.

Results and discussion

Method development

Our study aimed to design a quick and effective screening method to distinguish isomeric MA and PTM utilizing the advantages of combined FIA and MRM³ technique. Under positive ESI mode, both MA and PTM were found to form the [MH] ⁺ adducts and had the same nominal mass of 150.1 m/z. Upon MS/MS fragmentation, both compounds had comparable product ions. At least two SRM transitions for relative abundance ratio matching are utilized in routine confirmation and quantification method.^19,20,22 In the present case, SRM combinations for MA will also coincide with SRM for PTM due to similar fragment ions. Moreover, the quantification trace 150 > 91 gives a high-count signal regardless of either single spiked MA and/or PTM in the sample. Further disadvantage in the FIA paired with conventional MRM method includes the presence of substantial MA signal at 150 > 119 m/z even in blank specimens spiked with PTM. In the FIA-MRM³ technique, the MRM³ selectivity towards targeted transitions has additional identification dimension to substitute the absence of retention time information. Furthermore, similar concept of MS³ technique was described with the purpose of library matching using the scanned mass ranges of the second product ions.²³ Thus, the qualifying transitions, that is, 150 > 119 and 150 > 133, were chosen as the differentiation criteria in determining the presence of the isomeric MA and PTM confidently.

The endogenous matrix interferences and trace concentration was a concern for practical adoption reason. The sequential delivery of samples into the mass spec via FIA was observed to increase the background signal noise level and could introduce interferences that bear similar MRM transitions. Moreover, sample with low concentration risk having false negative result due to the monitored qualifier transition signals falling below the threshold level. The effect of biological matrix interferences in MA and PTM screening methodologies were also reported in previous enantiomer studies.^2,6

Interestingly, the developed FIA-MRM³ method did not show compromised results for all the sample sets and performed well with the developed technique (Figure 1). In the MRM³ method, the selectivity was enhanced due to the reduced probability of common product ions occurrence. Additional selection criterion towards the fragmented second precursor ions effectively eliminates the matrix interferences since monitored product transition for interfering ions are different from that of target analyte. In previous studies, MS³ ^23,24 and MRM³ ^25,26 were performed in tandem with MRM acquisition method to support retrospective analysis. The developed FIA-MRM³ method offers high-throughput information for screening purposes for large sample quantity before submitting to chromatographic-MS acquisitions. Such screening method cuts analysis time up to 80% and saves on solvent consumptions as well as prolonging column lifetime without compromising on sensitivity and selectivity. The actual confirmation and quantification method can then be proceeded for the positive samples only.

Figure 1.

(a, b) MRM³ collected spectra for MA and PTM standards spiked in whole blood sample. MA: methamphetamine; PTM: phentermine.

Linearity, LOD, LOQ and carry over

A standard curve of six points was constructed by determining the best fit of peak height ratios (peak height ratio of the analyte to internal standard) versus the analyte concentration with linear regression of 1/x weightage. Dynamic linear ranges, LOD, LOQ and calibration results were shown in Table 3. The results showed good linear relationships with correlation coefficients greater than 0.990 for all the targeted analytes.

Table 3.

Calibration curve dynamic range and compound extraction protocol LOD and LOQ.

Analyte	LOD (ng/mL)	LOQ (ng/mL)	Slope	Intercept	R ²
Whole blood
MA	2.23	7.45	20,794.30	20,103.02	0.994
PTM	2.07	6.90	19,752.60	265.73	0.995
DBS
MA	3.40	11.33	20,200.99	21,896.81	0.990
PTM	2.86	9.53	22,074.71	62,103.69	0.990

LOD: limit of detection; LOQ: limit of quantification; MA: methamphetamine, PTM: phentermine; DBS: dried blood stain.

No analyte was detected in both blank sample and blank solvent injected immediately following analysis of 200 ng/mL calibrator and after each of five replicates injection of 100 ng/mL in a batch run indicating the absence of carry over effect.

Accuracy and precision

Method validation results for accuracy and precision are summarized in Table 4. The recoveries for both tested analytes were all within +20% error while the intra-day and inter-day precision results have shown acceptable precision of which all were less than 10% RSD. The results revealed good precisions and accuracies as well as established the robustness of the method.

Table 4.

Accuracy and precision.

Analyte	Conc. (ng/mL)	Mean (ng/mL)	Accuracy (%)	Standard deviation	CV%
Whole blood
MA	20	20.00	99.97 ± 16.6	3.33	9.34
	100	100.03	100.03 ± 8.1	8.12	9.01
PTM	20	19.99	100.02 ± 19.7	3.93	9.55
	100	99.96	99.96 ± 15.1	10.06	9.55
DBS
MA	20	22.27	111.19 ± 27.7	5.08	7.17
	100	100.03	100.06 ± 27.8	9.08	9.07
PTM	20	19.99	105.11 ± 22.1	7.86	7.98
	100	99.99	108.59 ± 24.8	10.38	8.40

MA: methamphetamine; PTM: phentermine; CV: coefficient of variation; DBS: dried blood stain.

Selectivity and matrix effects

A typical chromatogram for the double blank control (free of analytes and internal standard) is shown in Figure 2. No interfering peaks from endogenous compounds were observed at the retention times of the analytes or the internal standard from the blank matrix. While Figure 3 showed the presence of both analytes (MA and PTM) with total data acquisition time of 2 min. Acceptance criteria for % matrix effect are set at 75–125% with a coefficient of variation (% CV) of maximum 15%.²⁷

Figure 2.

Representative chromatograms (a) double blank (tested for MA) and (b) double blank (tested for PTM). MA: methamphetamine; PTM: phentermine.

Figure 3.

Cross-analyte examination of MA and PTM with MS³ library matching. MA: methamphetamine; PTM: phentermine.

Matrix effect was evaluated as per Matuszewski et al.²⁸ equation

% Matrix effect = \frac{Response post−extracted spiked sample}{Response non−extracted neat sample} × 100

where the post-extracted spiked sample contains the analytes added into the blank blood matrix. The non-extracted neat sample contains the analytes added to the mobile phase (0.1% formic acid in water:0.1% formic acid in acetonitrile). This was performed in replicates of seven (n = 7) and the results were 80–110% with CV less than 10% for the both analytes. Addition of the isotopically labelled internal standard of MA-d₁₄ coupled with the MRM³ with unique identification and highly selective mode, helped in reducing the matrix effects. The observed reduced matrix effect would be helpful for the differentiation of isomeric compounds and quantification of the compounds for the desired forensic application.

Cross-analyte examination

For additional investigation, the presence of cross-analyte was evaluated since separation between the isomers was not performed in the FIA and the identification of analytes was entirely based on the strength of the MRM³ mode. The examination was performed using MultiQuant Software to cross-check the analyte in respective spiked sample. The results showed that PTM was not present in the singly MA spiked sample and vice versa (Figure 3) confirming that no cross-analyte occurred during analysis, and thus eliminating the possibility of false positive results.

Application on forensic toxicology cases in Malaysia

The developed and validated method was applied to whole blood and DBS as well as the PT samples. The laboratory responses and intended responses (previous cases samples) were in good agreement and are listed in Table 5. The results were subjected to qualitative analysis for the purpose of discriminating the isomeric MA and PTM in monitoring scheme.

Table 5.

Intended and laboratory response for MA and PTM in whole blood sample (WBS; n = 20) and DBS (n = 20).^a

Blood (n = 20)WBS and DBS	Intended response	Laboratory response
1	PTM	PTM
2	PTM	PTM
3	MA	MA
4	MA	MA
5	MA	MA
6	MA	MA
7	MA	MA
8	PTM	PTM
9	PTM	PTM
10	MA	MA
11	MA	MA
12	MA	MA
13	PTM	PTM
14	PTM	PTM
15	PTM	PTM
16	MA	MA
17	MA	MA
18	MA	MA
19	PTM	PTM
20	PTM	PTM
QC 1	MA	MA
QC 2	MA	MA
QC 3	MA	MA

MA: methamphetamine; PTM: phentermine; DBS: dried blood stain.

^aQuality control: College of American Pathologists (CAP), USA.

Conclusion

In conclusion, the developed method has been successfully demonstrated and applied to real case samples. The MRM³ technique has added another dimension in the determination of drugs in DBS thru the second generation of product ion. MRM³ in combination with the versatile FIA technique has successfully identified and confirmed the identity of isomeric compounds of similar masses in the simplest, economic and rapid analysis, which can be applied as routine analysis for application such as forensic toxicology.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Fathiah A Zubaidi

References

Pharmaceutical Services Division Ministry of Health Malaysia. Malaysian Drug Code. 6th ed. Malaysia: Ministry of Health, 2010, http://apps.who.int/medicinedocs/documents/s18584en/s18584en.pdf

Ward

Enders

Bell

. Improved chiral separation of methamphetamine enantiomers using CSP-LC–MS-MS. J Anal Toxicol 2016; 40(4): 255–263.

Mbughuni

Jannetto

Langman

. Mass spectrometry applications for toxicology. EJIFCC 2016; 27(4): 272–287.

Maurer

. What is the future of (ultra) high performance liquid chromatography coupled to low and high resolution mass spectrometry for toxicological drug screening? J Chromatogr A 2013; 1292: 19–24.

Arnhard

Gottschall

Pitterl

. Applying “Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra” (SWATH) for systematic toxicological analysis with liquid chromatography-high-resolution tandem mass spectrometry. Anal Bioanal Chem 2015; 407(2): 405–414.

Adamowicz

Kala

. Enantioselective method for determination of amphetamine and its eight analogues using chiral stationary phase. Prob Forensic Sci 2000; 44: 24–42.

Maurer

. Advances in analytical toxicology: the current role of liquid chromatography–mass spectrometry in drug quantification in blood and oral fluid. Anal Bioanal Chem 2005; 381(1): 110–118.

Buse

Badea

Verrall

. A general liquid chromatography tandem mass spectrometry method for the quantitative determination of diquaternary ammonium Gemini surfactant drug delivery agents in mouse keratinocytes’ cellular lysate. J Chromatogr A 2013; 1294: 98–105.

Gorlach

Richmond

. MipTec proceedings: high-throughput flow injection analysis mass spectroscopy with networked delivery of colour rendered results. JALA: Journal of the Association for Laboratory Automation 1998; 3(4): 56–57.

10.

Marin

Doyle

Chang

. One hundred false-positive amphetamine specimens characterized by liquid chromatography time-of-flight mass spectrometry. J Anal Toxicol 2015; 40(1): 37–42.

11.

Cody

. Determination of methamphetamine enantiomer ratios in urine by gas chromatography–mass spectrometry. J Chromatogr 1992; 580(1–2): 77–95.

12.

Johnson

. An acid hydrolysis method for quantification of plasma free and total carnitine by flow injection tandem mass spectrometry. Clin Biochem 2010; 43(16–17): 1362–1367.

13.

Sander

Becker

Thiery

. Simultaneous identification and quantification of triacylglycerol species in human plasma by flow-injection electrospray ionization tandem mass spectrometry. Chromatographia 2015; 78(5-6): 435–443.

14.

Niesser

Koletzko

Peissner

. Determination of creatinine in human urine with flow injection tandem mass spectrometry. Ann Nutr Metab 2012; 61(4): 314–321.

15.

Míguez Framil

Moreda-Piñeiro

Bermejo-Barrera

. Electrospray ionization tandem mass spectrometry for the simultaneous determination of opiates and cocaine in human hair. Anal Chim Acta 2011; 704(1–2): 123–132.

16.

John

Eddleston

Clutton

. Simultaneous quantification of the organophosphorus pesticides dimethoate and omethoate in porcine plasma and urine by LC–ESI-MS/MS and flow-injection-ESI-MS/MS. J Chromatogr B 2010; 878(17-18): 1234–1245.

17.

Michel

Gaunt

Arnason

. Development and validation of fast and simple flow injection analysis–tandem mass spectrometry (FIA–MS/MS) for the determination of metformin in dog serum. J Pharm Biomed Anal 2015; 107: 229–235.

18.

Jurdakova

Górová

Addová

. FIA-MS/MS determination of creatinine in urine samples undergoing butylation. Anal Biochem 2018; 549: 113–118.

19.

Scientific Working Group for Forensic Toxicology (SWGTOX). Standard practices for method validation in forensic toxicology. J Anal Toxicol 2013; 37(7): 452–474. DOI: 10.1093/jat/bkt054.

20.

Drugs UNODC Laboratory and Scientific Section. Guidance for the validation of analytical methodology and calibration of equipment used for testing of illicit drugs in seized materials and biological specimens: a commitment to quality and continuous improvement. New York: United Nations Publications, 2009.

21.

Winek

Wahba

Winek

Jr . Drug and chemical blood-level data 2001. Forensic Sci Int 2001; 122(2): 107–123.

22.

European Commission Directorate-General for Health and Food Safety. Guidance document on analytical quality control and method validation procedures for pesticide residues and analysis in food and feed (SANTE 11813), 2017, p. 46, https://www.accredia.it/app/uploads/2015/12/6215_plant_pesticides_mrl_guidelines_wrkdoc_11945_en.pdf

23.

Cesari

Fontana

Montanari

. Development and validation of a high-throughput method for the quantitative analysis of d-amphetamine in rat blood using liquid chromatography/MS3 on a hybrid triple quadrupole-linear ion trap mass spectrometer and its application to a pharmacokinetic study. J Chromatogr B 2010; 878(1): 21–28.

24.

Hopfgartner

Varesio

Tschäppät

. Triple quadrupole linear ion trap mass spectrometer for the analysis of small molecules and macromolecules. J Mass Spectrom 2004; 39(8): 845–855.

25.

Wright

Thomas

Stanford

. Multiple reaction monitoring with multistage fragmentation (MRM3) detection enhances selectivity for liquid chromatography-tandem mass spectrometry analysis of plasma free metanephrines. Clin Chem 2015; 61: 505–513.

26.

Schmidlin

Garrigues

Lane

. Assessment of SRM, MRM3, and DIA for the targeted analysis of phosphorylation dynamics in non-small cell lung cancer. Proteomics 2016; 16(15–16): 2193–2205.

27.

Remane

Meyer

Peters

. Fast and simple procedure for liquid–liquid extraction of 136 analytes from different drug classes for development of a liquid chromatographic-tandem mass spectrometric quantification method in human blood plasma. Anal Bioanal Chem 2010; 397(6): 2303–2314.

28.

Matuszewski

. Standard line slopes as a measure of a relative matrix effect in quantitative HPLC–MS bioanalysis. J Chromatogr B 2006; 830(2): 293–300.