Validated LC-MS/MS method for simultaneous quantification of serotonin,kynurenine,and tryptophan in feline and canine plasma

Abstract

Tryptophan metabolites, such as serotonin and kynurenine, are neurotransmitters that play crucial roles in regulating mood, sleep, and various physiologic functions. We developed and validated a robust and sensitive method for the simultaneous quantification of serotonin (5-HT), kynurenine (KYN), and tryptophan (TRP), in feline and canine plasma using electrospray ionization liquid chromatography-tandem mass spectrometry (ESI-LC/MS/MS). Plasma samples underwent protein precipitation followed by chromatographic separation on a reversed-phase C18 column under isocratic elution. The method was validated, assessing linearity, accuracy, precision, recovery, and matrix effects in both cat and dog plasma. The method had high sensitivity, with limits of detection of 0.2 ng/mL for 5-HT, 0.08 ng/mL for KYN, and 2.5 ng/mL for TRP, and limits of quantification of 0.35, 0.22, and 4.2 ng/mL, respectively. Calibration curves showed excellent linearity (R² > 0.995) across biologically relevant concentration ranges. Intra- and inter-day accuracy and precision were within acceptable limits (<15% relative SD). The method had good recovery and minimal matrix effects. The method was further applied to plasma samples from healthy dogs and cats to demonstrate its applicability to biological samples and to report analyte concentrations in clinically healthy animals. Our validated LC-MS/MS method enables precise and reliable quantification of 5-HT, KYN, and TRP in feline and canine plasma.

Keywords

canine plasma feline plasma kynurenine LC-MS/MS serotonin tryptophan

Tryptophan (TRP) is a precursor of several important biological pathways, such as protein biosynthesis and metabolic network regulation. Ingested TRP is mainly metabolized via the kynurenine (KYN) and the serotonin (5-hydroxytryptamine, 5-HT) pathways in mammalian species.³ TRP, KYN, and 5-HT play pivotal roles in mammalian physiology, particularly in maintaining normal brain function,^18,21,24,27 immune system function,^17,19 and regulating gastrointestinal health and homeostasis.^4,9,22 For instance, chronic inflammatory enteropathy in cats was associated with decreased TRP and decreased microbial indole catabolites of TRP.⁴ In dogs, an association was found between reduced serum 5-HT concentrations and increased aggressive¹⁸ or attention-deficit/hyperactivity disorder¹³–like behaviors.

Measurements of TRP, KYN, and 5-HT in mammalian biological samples have often been performed using high-performance liquid-chromatography (HPLC) coupled to various methods of detection such as electrochemical detection (HPLC-EC),¹⁰ fluorescence detection,^12,25 and UV absorbance.²⁹ Besides chromatographic methods, commercial ELISAs have also been used.^20,24 However, these techniques are limited because of low sensitivity and specificity (because of interference with other compounds in the matrix) and are limited to single-analyte measurement.²⁶ For instance 5-HT levels were evaluated in equine plasma using both LC-MS/MS and 5-HT-ELISA³⁰; Passing–Bablok regression and Bland–Altman analysis revealed poor agreement between the 2 assays.

To address these limitations, mass spectrometry (MS) has become a key mode of detection of TRP and its metabolites because of its high degree of selectivity, specificity, and sensitivity. Numerous liquid chromatography–tandem MS (LC-MS/MS) methodologies have been developed to quantify TRP, KYN, and 5-HT, with TRP detection methods extensively summarized in a comprehensive review.²⁶ The high endogenous TRP and KYN concentrations in plasma do not require a highly sensitive methodology; however, the measurement of 5-HT requires a highly sensitive technique, given that endogenous levels have been observed at low ng/mL concentration. Analysis using LC-MS has been performed extensively, mainly in human samples, using different extraction protocols and solvent combinations ( Suppl. Table 1 ). However, the reliability of the available methods in companion animals, particularly cats and dogs, remains unknown.

To our knowledge, no LC-MS/MS method has been developed and validated to measure simultaneously TRP, KYN, and 5-HT in canine and feline plasma. We retrieved no reports in a search of Google Scholar, PubMed, Jisc Library Hub Discover, Web of Science, using the search terms “tryptophan”, “kynurenine”, “serotonin”, “plasma”, “canine”, “feline”, AND “LC-MS/MS,” and related keywords.

Our first aim was to develop and validate a sensitive method for the simultaneous analysis of TRP, KYN, and 5-HT using LC-MS/MS, with stable isotope-labeled internal standards (SIL-IS) for each compound, to reduce assay variation. We used a surrogate matrix for calibration curve and quality control samples, given the limited availability of a true blank matrix and the ethical need to ensure replacement, reduction, refinement, and responsibility (3Rs) of animal use.¹⁴ Our second aim was to describe the physiologic ranges of corresponding concentrations in plasma of healthy domestic dogs and domestic shorthair cats.

Materials and methods

Chemicals and reagents

All reagents used were of analytical grade: TRP (>98%), KYN (>98%), 5-HT (>97%), d4-5-HT HCl (100 µg/mL); bovine serum albumin (BSA; 7.5% w/v in Dulbecco PBS [DPBS], Merck, Germany); 2H₅-L-TRP (99.9%) and [¹³C₆]-KYN (97.3%; Alsachim, France); acetonitrile (ACN) and formic acid (FA; LC/MS grade, Fisher, UK); ultrapure water (18.2 mΩ) via a Purite water purification system (Suez Water Purification Systems, UK).

Standard solutions

Stock standard solutions of target analytes and internal standards were prepared in ultra-pure water at 1,000 mg/mL for TRP, KYN, and 5-HT. Respective SIL-IS stock solutions were prepared at 1 mg/mL (2H₅-L-TRP, [¹³C₆]-KYN) and 2 mg/mL (d₄-5-HT HCl). Working solutions were prepared weekly at 10, 1, 0.1, 0.01, and 0.001 µg/mL for each target analyte. A global SIL-IS working solution was prepared in ultra-pure water at a final concentration of 10 µg/mL for 2H₅-L-TRP and [¹³C₆]-KYN, and 0.10 µg/mL for d₄-5-HT HCl. BSA (7.5% w/v in DPBS) was used as surrogate matrix for the preparation of QC samples because of the unavailability of true blank matrix.

Calibration curves

The calibration curve standards were prepared in water because a truly blank matrix (plasma) was unavailable, given the endogenous nature of the targeted analytes. Ten calibration standards were used at the levels 1, 2, 10, 20, 30, 40, 70, 90, 200, and 300 ng/mL for KYN. For 5-HT, this was 0.35, 1, 2, 4, 5, 7, 10, 20, 50, and 150 ng/mL. For TRP, this was 10, 20, 100, 160, 200, 300, 400, 1,000, 1,500, and 2,200 ng/mL. Additions of SIL-IS global solution contained 2H₅-L-TRP and [¹³C₆]-KYN at 200 ng/mL, and d₄-5-HT HCl at 2 ng/mL, final concentration. A blank sample of water and SIL-IS was applied to confirm lack of signal ( Fig. 1 ). A double blank (water, process double blank) without target analytes or SIL-IS was included in the analytical run.

Figure 1.

Extracted ion chromatograms of blanks spiked with stable isotope-labeled internal standards (SIL-IS): A) 5-HT; B) KYN; C) TRP; D) 2H₅-L-tryptophan; E) [¹³C₆]-kynurenine; F) deuterated serotonin (5-HT-d₄).

Sample preparation

Frozen plasma samples were thawed and then vortexed to ensure homogeneity. For each sample, 30 μL of plasma, BSA (7.5% w/v in DPBS QC samples), or water (for process blanks) was transferred to a 1.5-mL tube (Protein LoBind; Eppendorf). Subsequently, 62 μL of 0.1% formic acid in acetonitrile (v/v) and 9 μL of global SIL-IS working solution was added. For QC samples, the vials were spiked with appropriate working solutions containing the 3 analytes ( Table 1 ). The mixtures were sonicated in a cold-water bath for 5 min and then centrifuged (16,500 × g for 10 min at 4°C). After centrifugation, 15:85 0.1% formic acid ACN:water (v/v) was slowly added to each tube to bring the final volume to 450 μL. Samples were then centrifuged (16,500 × g for 1 min at 4°C), and the supernatant was filtered using a 0.22-µm microporous polytetrafluoroethylene (PTFE) membrane. The filtered supernatant was transferred to vials with inserts and samples stored at −80°C for up to 24 h before LC-MS/MS analysis.

Table 1.

Intra-batch accuracy and precision, and inter-batch accuracy and precision, using liquid chromatography–mass spectrometry.

Analyte/nominal concentration, ng/mL	Day 1			Day 2			Day 3			Inter-day
Analyte/nominal concentration, ng/mL	Measured concentration, $\bar{x}$ (SD)	Accuracy*, %	Precision†, %	Measured concentration, $\bar{x}$ (SD)	Accuracy, %	Precision, %	Measured concentration, $\bar{x}$ (SD)	Accuracy, %	Precision, %	Accuracy‡, %	Precision§, %
5-HT
1	1.1 (0.1)	108	5.8	1.1 (0.1)	105	6.8	0.9 (0.1)	94.1	11.3	101	10.3
4	3.8 (0.2)	94.1	4.2	4.1 (0.1)	102	3.5	3.8 (0.2)	94.0	6.1	96.1	6.0
10	10.5 (1.1)	106	10.0	9.8 (0.5)	98.5	5.4	9.5 (0.3)	94.9	3.3	98.7	7.3
50	56.9 (0.7)	114	1.2	50.3 (5.1)	101	10.1	48.7 (3.7)	97.4	7.6	103	9.5
KYN
1	1.0 (0.1)	96.0	6.1	1.0 (0.1)	105	6.1	1.1 (0.1)	106	9.6	103	8.4
20	19.0 (0.5)	95.0	2.7	19.5 (1.0)	97.9	5.0	18.1 (0.7)	90.7	3.7	93.9	4.7
70	71.4 (2.3)	102	3.2	70.3 (1.5)	103	2.1	65.6 (1.0)	93.7	1.5	98.5	4.5
200	200 (0.6)	100	0.3	203 (1.4)	101	0.7	193 (3.4)	96.4	1.8	98.7	2.6
TRP
20	22.1 (0.7)	111	3.2	21.9 (1.4)	110	6.5	18.7 (1.0)	93.3	5.2	103	9.7
160	159 (3.5)	99.3	2.2	160 (2.3)	99.8	1.5	157 (5.1)	98.1	3.3	98.9	2.5
400	386 (4.3)	96.5	1.1	410 (9.6)	102	2.3	388 (14.5)	97.1	3.7	98.3	3.7
1,500	1.36 × 10³ (14.1)	90.9	1.0	1.45 × 10³ (18.9)	96.7	1.3	1.50 × 10³ (34.8)	100	2.3	96.7	4.5

Intra-day accuracy was the amount of target analytes added to a blank surrogate matrix (7.5% w/v BSA in DPBS), carried throughout the extraction procedure, and then analyzed. The % accuracy was based on the mean of 3 samples at 4 concentration levels.

†

Intra-day precision was the amount of target analytes added to a blank surrogate matrix (7.5% w/v BSA in DPBS), carried throughout the extraction procedure, and then analyzed. The precision was evaluated as CV based on the mean of 3 samples at 4 concentration levels.

‡

Inter-day accuracy was the average of 3 samples, obtained in days 1–3, at each concentration level.

Inter-day precision was the amount obtained from the CVs. The inter-day precision % is the average % precision obtained in days 1–3.

Instrumental analysis

Chromatographic separations were performed on an LC system (1260 Infinity; Agilent). Chromatographic separation was achieved on a column (3.0 mm i.d. × 150 mm, 2.7 µm; Supelco Ascentis Express C18, Millipore Sigma) equipped with a corresponding guard-column (2.7 μm; Ascentis Express 90 Å C18, Millipore Sigma) and was an adaptation from 2 methods.^8,31 The aliquots of samples were eluted under isocratic conditions over 4 min at a flow rate of 0.5 mL/min. The mobile phase was composed of 0.1% formic acid in 15:85 ACN:water (v/v). Column temperature was maintained at 25°C. To reduce the cleaning time of the ion source, a valve was used to divert to “off-line” waste for the first 1 min and for the last 1 min of analysis. An injection volume of 4 µL was used. Samples were analyzed by a mass spectrometer (Triple Quad 6460; Agilent) coupled with electrospray ionization (Jet Stream technology; Agilent) using positive electrospray ionization and scheduled multiple reaction monitoring (MRM) mode. Software (v.B.04.01, MassHunterData Acquisition; Agilent) was used to control the equipment. Settings were as follows: capillary voltage, +4 kV; nozzle voltage, +500 V. Nitrogen was applied as a nebulizer gas of 55 psi, a carrier gas of 13 L/min at 290°C, and a sheath gas of 12 L/min at 350°C. MRM transitions, collision energies (CE), and fragmentor voltages for all compounds were auto-optimized in positive mode by a workstation (Optimizer of MassHunter; Agilent) to enhance selectivity and sensitivity of the method ( Table 2 , Fig. 2 ).

Table 2.

Tandem mass spectrometry parameters and multiple reaction monitoring (MRM) transitions in positive mode.

Analyte	Retention time, min	MRM transition, m/z	Collision energy, V	Fragmentor, V
5-HT	1.26	177.1 → 160.0, 115.1	8, 28	68
5-HT-d₄	1.26	181.1 → 164.0, 136.1	8, 24	63
KYN	1.52	209.1 → 192.0, 146.0	0, 16	73
[¹³C₆]-KYN	1.52	215.1 → 198.0, 152.1	0, 16	73
TRP	1.86	205.1 → 188, 146	8, 16	78
2H₅-L-TRP	1.83	210.1 → 192.1, 150.1	8, 16	78

Product ions used for quantitation are in bold.

Figure 2.

Representative extracted ion chromatograms from a feline plasma sample: A) 5-HT and respective structure; B) KYN and respective structure; C) TRP and respective structure; D) 2H₅-L-tryptophan; E) [¹³C₆]-kynurenine; F) deuterated serotonin (5-HT-d₄).

Method validation

Calibration curves, LOD, and LOQ

The linearity range was determined by examining plasma samples from healthy animals: 3 dogs, 3 cats. Calibration curves were prepared in water and analyzed in triplicate. The peak area response ratios (analyte peak area/IS peak area) were calculated and plotted against their corresponding nominal concentrations. Linear regression was employed, with (1/X) used as a weighting factor. The linearity was deemed satisfactory if the coefficient of determination (R²) was >0.995. The limit of detection (LOD) was calculated as the analyte concentration giving a signal equal to the blank signal (process blank in water, n = 7), y_blank, plus 3 SDs of the blank signal, SD_yblank. LOD = y_blank + 3 SD_yblank. Limit of quantification (LOQ) was calculated as the analyte concentration giving a signal equal to the blank signal (process blank in water, n = 7) plus 10 SDs of the blank. LOQ = y_blank + 10 SD y_blank.

Matrix effects

Matrix effects were evaluated based on the methods described elsewhere.^11,15 Parallelism is generally understood to mean how well a set of calibration standards trails the response of the analyte of interest in the biological matrix.^16,31 Parallelism was assessed by comparing the slope of a calibration curve created in water and a separate calibration curve in BSA (7.5% w/v in DPBS), with a calibration curve constructed in spiked canine and feline plasma samples. The samples were prepared by spiking the different matrices with 9 concentrations of target metabolites. The samples were then prepared in triplicate, and the peak area ratio and nominal spike concentration were plotted. Then, a simple linear regression was performed, and parallelism was evaluated by statistically comparing the slopes of the curves.

Accuracy, precision, and recovery

Method accuracy and precision were determined in QC-BSA (7.5% w/v in DPBS) spiked with known amounts of target compounds (Table 2) and evaluated by measuring 3 replicates at 4 different concentrations on 3 different days. The accuracy was determined by the percentage of the nominal concentration. The precision was expressed by repeatability as CV%.

Inter-day and intra-day precision were calculated for all QC samples. Inter-day precision (CV%) = SD of $\bar{x}$ in 3 d/ $\bar{x}$ concentration in 3 d. Intra-day precision (CV%) = SD in 1 d/ $\bar{x}$ concentration in 1 d. The recovery (%) in plasma samples was determined by dividing the measured concentration after spiking by the sum of the endogenous and spiked concentrations, then multiplying by 100%.

The acceptance criteria for the CV was set at 15% and for the recovery at 70–130%.^2,7

Carry-over

Carry-over was evaluated by analyzing double blank samples following the analysis of the highest calibration standard (n = 5) and highest QC sample (n = 5). This was also completed randomly between feline and canine plasma samples (n = 5).

Stability

Processed sample stability was evaluated by injecting processed low and high QC samples immediately after creation, as well as processed samples stored at 5°C for 24, 48, or 72 h, and for 2 mo at −80°C. In addition, the pre-extraction stability of 2 feline and canine plasma samples was evaluated by separately aliquoting and directly storing for 1–60 d at −80°C. Analyses were all performed in triplicate. The analytes were considered stable when the CV% was <15%.

Feasibility test

The established method was applied to support veterinary studies to measure the plasma concentration of the 3 analytes in 54 healthy, adult dogs (1–11-y-old; 22 female, 32 male; 22 Labrador Retrievers, 19 Beagles, 13 Norfolk Terriers) and 26 healthy adult cats (3–12-y-old; 15 females, 11 males; domestic shorthair cats). Each blood sample was collected in the morning, placed in lithium heparin-coated tubes, and centrifuged at 2,000 × g for 10 min at 4°C within 2 h. Plasma was separated and then stored at −80°C for future analysis. The animal studies were approved by the Waltham Animal Welfare and Ethical Review Body (PPMs 125075, 54921). We conducted our study in accordance with local legislation and institutional requirements.

At the beginning of the run, a blank (water) and a standard solution containing a known concentration of the SIL-ISs (system suitability test) were injected in triplicate. A double blank (only water) and a process blank (water with SIL-IS) extraction was always performed in duplicate and analyzed in each run. A freshly prepared calibration curve was run each week (in triplicate). To keep track of the accuracy and precision, low (1 ng/mL for 5-HT, 1 ng/mL for KYN, and 20 ng/mL for TRP) and high (50 ng/mL for 5-HT, 200 ng/mL for KYN, and 1,500 ng/mL for TRP) QC samples were extracted and injected during the run and placed every 10 samples.

Data analysis

Integration of the chromatographic peaks corresponding to selected product ions generated from the precursor [M+H]⁺ ions, together with quantitation and data processing, were performed (v.B.05.01, MassHunter quantitative QQQ analysis; Millipore Sigma). Compounds were positively identified by matching ion ratios and retention times to those of reference standards, with acceptance criteria of ±20% for ion ratios and ±0.1 min for retention times.⁶

Calibration curves were constructed (v.B.05.01; MassHunter quantitative QQQ analysis) and R software (v.3.5 for Windows, https://www.r-project.org/). Statistical tests were conducted using R and Excel (v.16.0, 2021; Microsoft). The slopes and the CIs in the parallelism experiment were calculated by conducting a linear regression analysis. The differences between water and plasma slopes for each analyte were evaluated with a paired t-test. The criterion for statistical significance was p ≤ 0.05, after adjustment for multiple comparisons using Bonferroni correction.

Results

Linearity, limit of detection, and limit of quantification

The mean coefficient of determination (R²) for all the analytes was >0.995, indicating excellent linearities. The calibration ranges were sufficient to cover analyte concentrations in all feline and canine plasma samples ( Table 3 ).

Table 3.

Linear range, limit of detection (LOD), limit of quantification (LOQ), representative regression equations of targeted analytes, coefficient of determination (R²), and carry-over.

Analytical performance characteristic	5-HT	KYN	TRP
Linear range, ng/mL	0.35–150	0.22–300	10–2,500
LOD, ng/mL	0.2	0.08	2.5
LOQ, ng/mL	0.35	0.22	4.2
Representative regression equation	0.140x + 0.0211	0.0110x + 0.0015	0.0126x + 0.0017
Coefficient of determination, R²	0.996	0.997	0.998
Carry-over	Not higher than 3% of LLOQ	Not higher than 9% of LLOQ	Not higher than 0.8% of LLOQ

LLOQ = lower LOQ.

Matrix effects

For 5-HT, the slope differences between water/BSA and canine/feline plasma were statistically non-significant (p >0.2; Fig. 3 , Table 4 ).

Figure 3.

Evaluation of matrix effects, parallelism assessment. Curves obtained in water, BSA (7.5% w/v in DPBS), pooled canine plasma, and pooled feline plasma, spiked (n = 3) with the same amounts of target metabolites. Curves are shown for A) 5-HT, B) KYN, and C) TRP. X-axis: nominal concentration of spiked unlabeled metabolites. Y-axis: ratio (peak area of unlabeled metabolite/peak area of corresponding stable isotope-labeled internal standards).

Table 4.

Slopes of each curve performed on water, BSA (7.5% w/v in DPBS), canine and feline plasma, respective confidence intervals (CIs), and p-values for each slope comparison.

Matrix/group	Water	BSA	Canine plasma	Feline plasma	p-value
Matrix/group	Water	BSA	Canine plasma	Feline plasma	Watervs.canine plasma	BSAvs.canine plasma	Watervs.feline plasma	BSAvs.feline plasma
5-HT
Slope	1.26	1.13	1.22	1.20	0.721	0.262	0.617	0.721
95% CI	[1.23, 1.29]	[1.08, 1.19]	[1.14, 1.29]	[1.08, 1.30]
KYN
Slope	0.0118	0.0107	0.0121	0.0114	0.067	<0.001	0.067	<0.001
95% CI	[0.0115, 0.0121]	[0.0105, 0.0108]	[0.0119, 0.0124]	[0.0112, 0.0116]
TRP
Slope	0.0132	0.0126	0.0136	0.0142	0.11	<0.001	0.01	<0.001
95% CI	[0.013, 0.0135]	[0.0124, 0.0127]	[0.0132, 0.014]	[0.0136, 0.0149]

For KYN, the slope differences between water and canine/feline plasma were statistically non-significant (p = 0.067; Fig. 3, Table 4). However, the slope was significantly different (p <0.001) between BSA (7.5% w/v in DPBS) and canine and feline plasma and was ~6% and 12% lower in BSA (7.5% w/v in DPBS), respectively. Thus, for KYN, some matrix effects occurred, but were deemed acceptable.

For TRP, the slope differences between water and canine plasma were statistically non-significant (p = 0.11; Fig. 3, Table 4). However, the slope was ~7% lower in water than in feline plasma (p = 0.01). The slope of BSA (7.5% DPBS) was ~7% and 11% lower than in canine and feline plasma (p < 0.001), respectively. Therefore, TRP matrix effects were considered minor but acceptable.

Accuracy, precision, and recovery

Intra-day accuracy mean values were 94–114% for 5-HT, 91–105% for KYN, and 90.9–110.6% for TRP (Table 2). Inter-day accuracy was 96–103% for 5-HT, 94–102% for KYN, and 91–111% for TRP. Hence, the matrix effect in BSA (7.5% w/v in DPBS) was minimal when applying our calibration curve model and LC settings.

The recovery (%) in pooled feline and canine plasma was, respectively, 90–106% and 86–105% for 5-HT, 88–115% and 89–109% for KYN, and 96–113% and 88–104% for TRP ( Table 5 ).

Table 5.

Precision (CV%) and recovery coefficient in feline and canine pooled plasma.

Nominal spike concentration in pooled plasma	Feline (n = 4)			Canine (n = 4)
Nominal spike concentration in pooled plasma	Mean (SD)	CV%	Recovery SD (%)	Mean (SD)	CV%	Recovery SD (%)
Serotonin, ng/mL
X (endogenous)*	1.3 (0.1)	8.7	NA	5.9 (0.3)	5	NA
X + 1	2.3 (0.3)	12.3	101 (12.3)	7.0 (0.8)	11.0	102 (11.4)
X + 4	4.9 (0.6)	12.7	91.8 (11.6)	10.3 (0.5)	4.6	105 (4.8)
X + 5	6.7 (0.6)	8.3	107 (8.8)	11.9 (0.3)	7.6	109 (2.8)
X + 10	10.2 (1.4)	14	90.3 (12.6)	13.6 (1.0)	2.5	85.6 (6.5)
Kynurenine, ng/mL
X (endogenous)*	64.0 (0.3)	0.5	NA	70.8 (1.2)	1.7	NA
X + 1	65.1 (2.2)	3.3	100 (3.4)	78.1 (1.3)	1.6	108 (3.3)
X + 20	74.0 (3.8)	5.1	88.1 (4.5)	95.5 (0.6)	0.7	105 (0.7)
X + 30	108 (2.4)	2.3	115 (2.6)	89.8 (0.6)	0.2	89.1 (1.8)
X + 70	137 (2.7)	2	102 (2.0)	137 (0.2)	0.7	97.1 (0.8)
Tryptophan, ng/mL
X (endogenous)*	802 (11.2)	1.4	NA	682 (12.1)	1.8	NA
X + 20	838 (30.6)	3.7	102 (3.7)	728 (6.6)	0.9	104 (2.6)
X + 160	924 (12.7)	1.4	96 (1.3)	808 (1.9)	0.2	95.9 (1.2)
X + 200	1.13 × 10³ (13.2)	1.2	113 (1.3)	775 (5.7)	0.2	87.9 (1.3)
X + 400	1.31 × 10³ (24.4)	1.9	109 (2.0)	998 (1.7)	0.7	92.2 (1.2)

NA = not applicable; X = the endogenous concentration in the unspiked sample.

Endogenous values $\bar{x}$ (SD) are presented as n = 6. Values are reported as measured, without application of the dilution factor (DF = 15).

Carry-over

The carry-over observed was <9% of lower LOQ (LLOQ) for all analytes (Table 3).

Stability test

Surrogate matrix (7.5% w/v BSA in DPBS) spiked with target metabolites was stable at 5°C for 72 h, and at −80°C for 2 mo. No apparent change was observed in the concentrations of the feline and canine pool pre-processed samples at −80°C for 60 d.

Feasibility test

The plasma samples were run in 3 batches; accuracy and precision of the QC samples did not change significantly compared with the results of the method validation. Results are expressed as box plots with TRP, KYN, and 5-HT concentration (final concentration, after applying dilution factor) in the analyzed canine and feline plasma samples ( Fig. 4 ). Data from the feasibility test were expressed as median with interquartile range (IQR). The median TRP concentration in plasma for the feline cohort was 12.2 µg/mL (IQR 9.93–14.1; n = 26) and was 12.6 µg/mL (IQR 11.2–15.8; n = 54) for the canine cohort. The median KYN concentration in plasma for the feline cohort was 1.04 µg/mL (IQR 0.844–1.41; n = 26) and 0.948 µg/mL (IQR 0.680–1.23; n = 54) for the canine cohort. Regarding 5-HT, 15 dogs had levels below LOQ and were excluded from determining the median and IQR range. The median 5-HT concentration in plasma for the feline cohort was 20.9 ng/mL (IQR 9.0–77.6; n = 26) and 9.9 ng/mL (IQR 7.2–12.2; n = 39) for the canine cohort.

Figure 4.

Concentrations of tryptophan and its metabolites in plasma samples from 55 dogs and 20 cats. Plasma concentrations of A) 5-HT, B) KYN, and C) TRP. The center line is the median, the box is the interquartile range, and the whiskers are the 2.5th and 97.5th percentiles.

Discussion

A limitation of our study is that the target compounds are endogenous, making it difficult to obtain a truly “blank” matrix. To address this challenge, approaches such as standard addition, background subtraction, surrogate matrix, or surrogate analyte methods can be used.²⁸ We used a surrogate approach in water and BSA (7.5% w/v in DPBS) for the calibration curve and QC samples, respectively. The calibration curve in water and BSA (7.5% w/v in DPBS) was validated by checking parallelism with both canine and feline plasma. Minimal matrix effects were observed in both water and BSA (7.5% w/v in DPBS) matrices. Water/BSA are a quick and easy-to-obtain matrix for reproducible calibration and QC samples. Calibrators in water matrix provide a clean and consistent environment for linear calibration, helping to keep the LC-MS/MS system cleaner over time. Incorporating internal standards during both sample preparation and instrumental analysis is a widely adopted strategy to address analyte losses or matrix effects, particularly during electrospray ionization.²³ The application of SIL-IS effectively mitigates these challenges by stabilizing the analyte-to-IS signal ratio, regardless of variations in their absolute signal intensities.

The main advantages of this approach were its straightforward sample preparation through direct protein precipitation, minimal plasma requirement (30 μL), and short analysis time (4 min). The low sample volume, with BSA (7.5% w/v in DPBS) and water as surrogate matrices, adheres to the 3R ethical framework, helping minimize animal usage, while maintaining acceptable variability. Moreover, the demonstrated stability of pre-extracted plasma at −80°C for ≥2 mo supports the feasibility of long-term sample storage. The feasibility test verified that the calibration levels applied for all analytes were appropriate for plasma analysis of the canine and feline cohorts.

Comparable concentration ranges for TRP and KYN were observed across the feline and canine groups. Previous studies have reported similar TRP levels in canine plasma¹⁰ (~16,000 ng/mL) using HPLC-fluorometric detection; however, no such data are available for KYN nor feline plasma, with existing reports limited to serum.¹⁷ A wider IQR was observed for 5-HT levels in the feline cohort compared with the canine cohort, which may reflect species-specific physiologic differences. Higher concentration ranges for 5-HT have been previously reported in feline plasma using HPLC-ECD²⁴ (~430–1,100 ng/mL), compared with canine plasma¹⁸ (~20 ng/mL) using serotonin-ELISA methods. These differences may be attributed to factors such as breed, age range, pre-analytical variables (e.g., handling, storage time, temperature)^1,24 or the various sensitivity and matrix interferences inherent to these analytical techniques.^5,26 Because absolute concentrations can vary depending on experimental conditions and analytical techniques, direct cross-study comparisons are not advisable.³⁰ Therefore, establishing method-specific RIs for plasma TRP metabolites is recommended. It is important to note that, before measuring and reporting TRP metabolites concentrations in species lacking established analytical methods, researchers should evaluate and document the reliability of their techniques rather than assume their validity based on human data.

Supplemental Material

sj-pdf-1-vdi-10.1177_10406387261447261 – Supplemental material for Validated LC-MS/MS method for simultaneous quantification of serotonin, kynurenine, and tryptophan in feline and canine plasma

Supplemental material, sj-pdf-1-vdi-10.1177_10406387261447261 for Validated LC-MS/MS method for simultaneous quantification of serotonin, kynurenine, and tryptophan in feline and canine plasma by Carolina Maia, Morgan Farr, Sophia Dowell, Tim Finnis and Hannah Flint in Journal of Veterinary Diagnostic Investigation

Footnotes

Declaration of conflicting interests

All authors were employed by Mars Petcare, a manufacturer of pet food and provider of veterinary services.

Funding

All funding required for our study was provided by Mars Petcare.

Generative AI statement

The authors declare that no Gen AI was used in the creation of this manuscript.

ORCID iDs

Carolina Maia

Sophia Dowell

Tim Finnis

Hannah Flint

Supplemental material

Supplemental material for this article is available online.

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