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Analytic validation of a gas chromatography–mass spectrometry method for quantification of six amino acids in canine serum samples

Rosana LopesGastrointestinal Laboratory, Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843.

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Niels GrütznerGastrointestinal Laboratory, Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843.

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Nora BerghoffGastrointestinal Laboratory, Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843.

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Jonathan A. LidburyGastrointestinal Laboratory, Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843.

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Jan S. SuchodolskiGastrointestinal Laboratory, Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843.

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Jörg M. SteinerGastrointestinal Laboratory, Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843.

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Abstract

OBJECTIVE To analytically validate a gas concentration of chromatography–mass spectrometry (GC-MS) method for measurement of 6 amino acids in canine serum samples and to assess the stability of each amino acid after sample storage.

SAMPLES Surplus serum from 80 canine samples submitted to the Gastrointestinal Laboratory at Texas A&M University and serum samples from 12 healthy dogs.

PROCEDURES GC-MS was validated to determine precision, reproducibility, limit of detection, and percentage recovery of known added concentrations of 6 amino acids in surplus serum samples. Amino acid concentrations in serum samples from healthy dogs were measured before (baseline) and after storage in various conditions.

RESULTS Intra- and interassay coefficients of variation (10 replicates involving 12 pooled serum samples) were 13.4% and 16.6% for glycine, 9.3% and 12.4% for glutamic acid, 5.1% and 6.3% for methionine, 14.0% and 15.1% for tryptophan, 6.2% and 11.0% for tyrosine, and 7.4% and 12.4% for lysine, respectively. Observed-to-expected concentration ratios in dilutional parallelism tests (6 replicates involving 6 pooled serum samples) were 79.5% to 111.5% for glycine, 80.9% to 123.0% for glutamic acid, 77.8% to 111.0% for methionine, 85.2% to 98.0% for tryptophan, 79.4% to 115.0% for tyrosine, and 79.4% to 110.0% for lysine. No amino acid concentration changed significantly from baseline after serum sample storage at −80°C for ≤ 7 days.

CONCLUSIONS AND CLINICAL RELEVANCE GC-MS measurement of concentration of 6 amino acids in canine serum samples yielded precise, accurate, and reproducible results. Sample storage at −80°C for 1 week had no effect on GC-MS results.

Abstract

OBJECTIVE To analytically validate a gas concentration of chromatography–mass spectrometry (GC-MS) method for measurement of 6 amino acids in canine serum samples and to assess the stability of each amino acid after sample storage.

SAMPLES Surplus serum from 80 canine samples submitted to the Gastrointestinal Laboratory at Texas A&M University and serum samples from 12 healthy dogs.

PROCEDURES GC-MS was validated to determine precision, reproducibility, limit of detection, and percentage recovery of known added concentrations of 6 amino acids in surplus serum samples. Amino acid concentrations in serum samples from healthy dogs were measured before (baseline) and after storage in various conditions.

RESULTS Intra- and interassay coefficients of variation (10 replicates involving 12 pooled serum samples) were 13.4% and 16.6% for glycine, 9.3% and 12.4% for glutamic acid, 5.1% and 6.3% for methionine, 14.0% and 15.1% for tryptophan, 6.2% and 11.0% for tyrosine, and 7.4% and 12.4% for lysine, respectively. Observed-to-expected concentration ratios in dilutional parallelism tests (6 replicates involving 6 pooled serum samples) were 79.5% to 111.5% for glycine, 80.9% to 123.0% for glutamic acid, 77.8% to 111.0% for methionine, 85.2% to 98.0% for tryptophan, 79.4% to 115.0% for tyrosine, and 79.4% to 110.0% for lysine. No amino acid concentration changed significantly from baseline after serum sample storage at −80°C for ≤ 7 days.

CONCLUSIONS AND CLINICAL RELEVANCE GC-MS measurement of concentration of 6 amino acids in canine serum samples yielded precise, accurate, and reproducible results. Sample storage at −80°C for 1 week had no effect on GC-MS results.

Amino acids play a key role in many metabolic pathways. Since the 1950s,1 considerable research has been undertaken to investigate the mechanisms by which amino acids are absorbed,2 metabolized,3,4 and degraded as well as their roles in intracellular signaling.5–7 However, the mechanisms by which amino acids and their intermediate metabolites influence physiologic and pathological processes are not completely understood. Serum amino acid concentrations can be affected by various factors, including diet composition, gastrointestinal absorption, and hepatic metabolism. In human medicine, changes in serum amino acid concentrations have been reported for patients with gastrointestinal diseases, such as inflammatory bowel disease,8 colorectal cancer,9,10 ulcerative colitis and Crohn disease,11–13 pancreatitis,14 and gastric malignancy.15 However, serum amino acid concentrations have not been extensively evaluated in dogs with gastrointestinal disease.

Amino acids and their metabolic intermediates have direct or indirect influences on the gastrointestinal system.16,17 Digestibility is a primary factor that affects bioavailability of amino acids. The intestinal mucosa benefits directly from amino acids in the diet, which are used as a source of energy, for protein synthesis, in nitric oxide and glutathione production, and in modulation for signaling pathways.18–20 For example, intragastric exposure to tryptophan and phenylalanine can increase gastric secretions in dogs and humans,20,21 as can intragastric exposure to glutamate in dogs.22 Another important function of glutamate is modulation of physiologic activities in the gastrointestinal system through ionotropic and metabotropic receptors.21,23 These receptors are involved in local mechanisms of immune defense, regulation of motility, and regulation of epithelial cell secretion in the gastrointestinal tract. Furthermore, glutamate is important in the synthesis of glutathione, arginine, and proline.24 Glycine reportedly regulates pro- and anti-inflammatory cytokines in mice with endotoxin-induced liver disease by decreasing the serum concentration of tumor necrosis factor-α and increasing the serum concentration of interleukin-10.25 Abnormal tryptophan and tyrosine serum concentrations have been identified in elderly people in association with diabetes mellitus, gastrointestinal cancer, autoimmune disease, and bacterial or viral infections.26,27

Several methods have been used to quantify amino acids in biological samples, including thin-layer chromatography, ion-exchange chromatography, reversed-phase high-pressure liquid chromatography, and others.28 Gas chromatography–electron ionization mass spectrometry is a sensitive analytic technique with resolution and reproducibility superior to that of other methods for measuring these small molecules.29 Use of MSTFA reportedly improves reproducibility and recovery of amino acids during analysis.30 To the authors’ knowledge, analytic validation of a method for measurement of amino acids in serum samples from dogs by use of GC-MS has not been reported. The objectives of the study reported here were to validate a GC-MS method for measurement of glycine, glutamic acid, methionine, tyrosine, tryptophan, and lysine concentrations in canine serum and to assess the stability of each amino acid in canine serum in various storage conditions.

Materials and Methods

Animals and serum samples

Two sets of serum samples were used for the study: one for validation of the GC-MS method, and the other for testing of amino acid stability in serum samples after storage. Surplus pooled serum from 80 serum samples from dogs of various health statuses submitted to the Gastrointestinal Laboratory at Texas A&M University were used for method validation. For evaluation of amino acid stability in serum samples, blood samples were collected for serum harvest from 12 healthy pet dogs of various breeds for which owner consent had been obtained. Four dogs were spayed females, and 8 were neutered males. Median age was 6.5 years (range, 1.3 to 11 years). Owners had reported no clinical signs of disease for their dogs, and no abnormalities were detected during physical examination of any dog. Additionally, no clinically important abnormalities were detected via serum biochemical analysis or gastrointestinal function tests (ie, serum cobalamin, folate, pancreatic lipase immunoreactivity, and trypsin-like immunoreactivity concentrations). The study protocol was approved by the Texas A&M University Institutional Care and Use Committee (protocol No. 2012-101).

Evaluation of the GC-MS method

Amino acid extraction and derivatization—Techniques used for amino acid extraction from serum samples and subsequent derivatization were similar to those described elsewhere,31 with a few modifications. All amino acid standards were purchased as powder (reagent grade > 98% purity) and were kept with desiccant in a tightly closed container to avoid humidity. Standard calibration curves and internal standards were prepared fresh daily and accurately measured by dry weight. Internal standards containing deuterium-labeled isotopes of each amino acid (glycine-d2,a dL-glutamic-d3 acid,b l-tryptophan-d5,c l-lysine-d4 hydrochloride,d l-methione-methyl-d3,e and l-tyrosine-d2f) were prepared at a concentration of 50 μmol/L each during the sample extraction procedure. Stock solutions of each nonisotopic amino acid (l-glycine, l-glutamic acid, l-methionine, l-tryptophan, l-tyrosine, and l-lysine)g were prepared at concentrations of 5,000 μmol/L each and diluted to final concentrations of 100, 80, 60, 40, 20, and 10 μmol/L, which were then used for calibration. Stock solutions for glycine, glutamic acid, methionine, and lysine were prepared by dissolving the amino acid in high-purity water and 0.1M hydrochloric acidg (pH, 1.0), whereas tryptophan and tyrosine were prepared in 1M sodium hydroxideg solution (pH, 14.0). Standard calibration curves were generated during the experiment to determine the relationship between instrument response and known concentrations of amino acid standards.

For amino acid extraction, 10 samples of surplus canine serum were thawed, 400-μL aliquots were transferred to 2-mL microcentrifuge tubes, and 200 μL of a mixture of the 6 deuterated amino acids was added to yield a final concentration of 50 μmol/L each. Finally, 1,200 μL of methanolg was added to each microcentrifuge tube. Serum samples were vortex mixed for 30 seconds and then centrifugedh at 15,800 × g at room temperature (21°C) for 15 minutes. After centrifugation, 370-μL aliquots of serum were transferred into 3 labeled microcentrifuge tubes and vacuumi dried at room temperature for 4 hours. Vacuum-dried serum samples were refrigerated for a maximum of 5 days before the derivatization procedure. After refrigeration, samples were vacuum dried for an additional 4 hours before derivatization to ensure complete dryness.

Derivatization of all 6 amino acids in 10 serum samples was performed by means of oximation (performed first to protect carbonyl groups) and silylation reactions. To obtain successful derivatization, a molar ratio of 4:1 was used to derivatize a total of 250μM of amino acids within the serum samples. Fifty microliters of methoxyamine-HClg in pyridine (20 mg/mL) was added to each microcentrifuge tube containing vacuum-dried serum, and the mixture was processed for 15 minutes in a heating block at 72°C to allow a complete oximation reaction. Subsequently, 100 μL of MSTFAg was added to each microcentrifuge tube, tube contents were vortex mixed, and tubes were returned to the heating block to process for an additional 15 minutes at 72°C. Afterward, microcentrifuge tubes were centrifuged at 15,800 × g at room temperature for 15 minutes, and the supernatant was harvested and analyzed via GC-MS.

GC-MS analysis—To separate the 6 amino acids from the microcentrifuge tube contents, 1 μL of each prepared serum sample was injected at a temperature of 280°C into a medium-polarity, low-bleed gas chromatography column.j A 4:1 split ratio mode was used, and helium was used as the carrier gas. The oven temperature was set at 70°C to start, with a hold time of 4 minutes, followed by a linear increase of 20°C/min until 300°C was reached, at which point the temperature was held for 4 minutes. A postrun period of 2 minutes was set at 300°C after each sample injection to remove any high-boiling matrix material remaining on the column. Oven temperature was allowed to cool to 70°C before the next injection. Transfer-line temperature was held at 240°C during the entire experiment.

Separated amino acids were analyzed over a run period of 19.5 minutes with a gas chromatographk and mass selective detector.l The mass spectrometer source was operated at a temperature of 250°C in electron ionization mode. Quantification of amino acids was performed by measuring the m/z for selected ions, which represents the amino acid distribution of ions by mass in serum samples and standards. Selected ion monitoring analysis was used to quantify all 6 serum amino acid concentrations. Amino acids and their deuterated isotopes were quantified by use of the ions at an m/z of 174 and 176 for glycine, 246 and 249 for glutamic acid, 176 and 179 for methionine, 179 and 181 for tyrosine, 231 and 236 for tryptophan, and 156 and 160 for lysine, respectively.

Results validation—The LOD for each of the 6 amino acids in canine serum samples was determined by dilutional parallelism. Six pooled serum samples were diluted 1:2, 1:4, and 1:8 with high-purity water. The limit of the blank of the assay was determined by measuring 6 replicates of blank samples for each amino acid that contained only the internal standards and calculating the mean and SD result, such that the limit of the blank was equivalent to the mean result for the blank plus 2 SDs.32 The LOD for each amino acid analyzed was determined by taking the limit of the blank and adding 2 times the SD computed from values for sample replicates (n = 6) with the lowest known sample concentration (ie, LOD = limit of the blank + 2[SDlow concentration sample]).32

Efficiency of amino acid extraction was determined by adding to 4 pooled serum samples known concentrations (high [200 to 75 μmol/L], intermediate [50 to 20 μmol/L], and low [10 to 5 μmol/L]) of amino acid standard solutions (ie, spiking concentrations). Recovery of spiking concentrations (ie, spiking recovery) for glutamic acid, glycine, and lysine was determined by use of 5 standard spiking concentrations ranging from 10 to 200 μmol/L. Spiking recoveries for methionine and tyrosine were evaluated by addition of 5 standard spiking concentrations ranging from 5 to 75 μmol/L. Spiking recovery for tryptophan was analyzed by use of 4 standard spiking concentrations ranging from 10 to 75 μmol/L. Extraction efficiency was evaluated for each amino acid as the percentage of spiking concentration recovered and was expressed as the ratio of observed values to expected values. Serum amino acid concentrations were analyzed by interassay runs on 6 days by use of 6 serum replicates for each amino acid with known spiked standard concentrations. Reproducibility of assay results was assessed by the percentage of spiking concentration recovered by calculation of CVs.33

Intra-assay precision was determined by analysis of 10 aliquots of pooled canine serum from 4 dogs, each analyzed in triplicate on the same day. Deuterated isotopes of amino acids were added as internal standards to each sample as previously described. Interassay analyses were determined by use of 10 aliquots of pooled canine serum from 8 dogs that were assayed on 6 consecutive days.

Stock standard solutions for each amino acid were prepared at a concentration of 5,000 μmol/L and diluted to 100, 80, 60, 40, 20, and 10 μmol/L. A calibration curve was plotted for each amino acid by measuring the area under the relevant peak of standard amino acid solution of known concentrations ranging from 10 to 100 μmol/L and of the deuterium-labeled internal standard.

Evaluation of amino acid stability in stored serum samples

Blood sample collection—Blood samples were collected from the 12 healthy dogs by jugular venipuncture with 1-inch, 20-gauge needles into 10-mL evacuated tubes.m Blood samples were allowed to clot for 30 minutes and then centrifuged at 1,500 × g for 15 minutes. Serum was harvested and separated into aliquots. Serum aliquots for baseline measurements of amino acid concentrations were used within 15 minutes after harvest, and the remaining serum was separated into 400-μL aliquots and immediately stored at 4°C, −20°C, or −80°C.

Stability evaluation—Measurement of baseline amino acid concentrations was performed by use of the described GC-MS method < 1 hour after blood sample collection. Stability of those amino acids in serum aliquots (ie, percentage of baseline amino acid concentration recovered) was determined after storage at the 3 temperatures for 24 hours, 7 days, 19 days, and 60 days after serum harvest.

Statistical analysis

A statistical software packagen was used for data analysis. Data were first assessed for a normal distribution by use of the Kolmogorov-Smirnov test. Amino acid stability data were then analyzed by use of repeated-measures 1-way ANOVA, followed by the Tukey test (for normally distributed data) or the Dunn test (for nonnormally distributed data).

Results

Evaluation of the GC-MS method

The LODs for the 6 amino acids in surplus pooled serum samples from 6 dogs as determined by dilutional parallelism ranged from 2.6 to 6.1 μmol/L. The lowest LOD was observed for glycine (LOD, 2.6 μmol/L) and the highest for lysine (LOD, 6.1 μmol/L; Table 1). Percentages of amino acid concentrations recovered by dilutional parallelism ranged from 77.8% to 123.0%.

Table 1—

Results of GC-MS quantification of 6 amino acid concentrations in samples of canine serum for tests of dilutional parallelism (ratio of observed to expected concentration; 6 replicates involving 6 pooled serum samples) and intra-assay and interassay variability (10 replicates involving 12 pooled samples).

Amino acidRatio of observed to expected concentration (%)Intra-assay CV (%)Interassay CV (%)Precursor ion m/zPrecursor ion retention time (min)LOD (μmol/L)
Glycine79.5–111.513.416.617410.32.6
Glutamic acid80.9–123.09.312.42467.82.7
Methionine77.8–111.05.16.31769.94.0
Tyrosine79.4–115.06.211.017912.15.3
Tryptophan85.2–98.014.015.123114.33.9
Lysine79.4–110.07.412.415611.46.1

Reproducibility of the GC-MS method, as verified by CVs for interassay variability in measurement of methionine, tyrosine, lysine, and glutamic acid, was 6.3%, 11.0%, 12.4%, and 12.4%, respectively. The CVs for tryptophan and glycine were 15.1% and 16.6%, respectively (Table 1).

Precision of the method, as determined by intraassay CVs for glutamic acid, lysine, methionine, and tyrosine, was 9.3%, 7.4%, 5.1%, and 6.2%, respectively. Intra-assay CVs for glycine and tryptophan were 13.4% and 14.0%, respectively (Table 1).

Satisfactory percentages of spiking concentration recovered (79.9% to 112.7%) were achieved for all 6 serum replicates to which known concentrations of the various amino acids had been added (Table 2).

Table 2—

Spiking recovery results for each of 6 runs of a GC-MS assay to detect amino acid concentrations in canine serum samples to which known concentrations of 6 amino acid standards were added.

Amino acidMean ± SD observed concentration (μmol/L)Concentration of added standard (μmol/L)Expected concentration (μmol/L)Ratio of observed to expected concentration (%)CV (%)*
Glycine12.3 ± 0.7NANANA5.8
 180.0 ± 14.6200212.384.88.1
 160.4 ± 18.9133145.3110.411.8
 66.4 ± 6.85062.3106.610.2
 36.2 ± 5.33345.379.914.7
 21.3 ± 1.91325.384.28.9
Glutamic acid8.3 ± 0.3NANANA3.5
 215.2 ± 3.7200208.3103.31.7
 159.2 ± 7.3133141.3112.74.6
 49.0 ± 3.55058.384.17.1
 36.1 ± 4.13341.387.311.4
 20.0 ± 1.41321.393.87.2
Methionine28.0 ± 2.4NANANA8.6
 100.3 ± 3.275103.097.43.2
 72.6 ± 4.15078.093.15.7
 47.6 ± 5.62048.099.111.8
 36.1 ± 3.01038.094.98.2
 30.8 ± 2.9533.093.29.4
Tyrosine11.8 ± 1.4NANANA12.2
 86.5 ± 5.97586.899.76.8
 61.3 ± 7.75061.899.212.6
 30.8 ± 2.32031.896.87.5
 22.7 ± 2.21021.8104.39.8
 17.2 ± 2.1516.8102.512.3
Tryptophan53.0 ± 5.3NANANA10.0
 113.8 ± 9.975128.088.98.7
 107.9 ± 1.950103.0104.81.8
 68.9 ± 6.82073.094.49.8
 66.8 ± 6.11063.0105.29.1
Lysine20.0 ± 2.6NANANA13.2
 240.7 ± 25.3200220.0109.410.5
 75.9 ± 4.17595.079.95.4
 62.7 ± 8.35070.0104.513.3
 38.9 ± 5.42040.097.313.8
 29.7 ± 1.91030.098.96.5

NA = Not applicable.

The CVs represent the percentage of spiking amino acid concentration recovered.

Evaluation of amino acid stability in stored serum samples

Results of stability testing of amino acids in stored serum samples from 12 healthy dogs were summarized (Tables 3–5). For samples stored at 4°C for 1 day, only serum lysine concentration differed significantly (P = 0.01) from that at baseline (immediately after samples were obtained). However, by 7 days of storage at the same temperature, a significant (P = 0.01) increase from baseline in serum glutamic acid and lysine concentrations was identified.

Table 3—

Mean ± SD amino acid concentrations in serum samples obtained from 10 healthy dogs measured immediately (baseline) or after storage at 4°C for 1 or 7 days with a GC-MS assay (30 runs/amino acid).

Amino acidBaselineDay 1Day 7P value*
Glycine21.2 ± 4.8a18.7 ± 4.7a19.3 ± 4.0a0.005
Glutamic acid3.4 ± 0.9a3.8 ± 0.8a4.6 ± 0.9b0.01
Methionine5.5 ± 1.2a5.6 ± 1.2a5.2 ± 1.1a0.009
Tyrosine6.8 ± 1.5a7.8 ± 2.1a7.0 ± 0.1a0.60
Tryptophan174.0 ± 68.5a179.0 ± 74.1a145.0 ± 63.8a0.006
Lysine24.8 ± 10.2a53.1 ± 22.1b53.1 ± 14.8b0.01

P value pertains to results of repeated-measures 1-way ANOVA.

Values with different superscript letters differ significantly (P < 0.05; post hoc test).

Table 4—

Mean ± SD amino acid concentrations in serum samples obtained from 5 healthy dogs measured immediately (baseline) or after storage at −20°C for 7 or 19 days with a GC-MS assay (15 runs/amino acid).

Amino acidBaselineDay 7Day 19P value
Glycine21.7 ± 6.9a21.3 ± 8.9a74.7 ± 12.3b0.01
Glutamic acid3.8 ± 1.1a3.2 ± 0.7a8.2 ± 1.7b0.01
Methionine5.0 ± 1.0a5.1 ± 1.1a4.9 ± 0.5a0.82
Tyrosine7.8 ± 1.5a8.9 ± 1.2a4.7 ± 0.6b0.01
Tryptophan196.0 ± 89.4a129.0 ± 49.3a140.1 ± 46.3a0.009
Lysine27.7 ± 12.9a53.3 ± 9.8b20.7 ± 5.6a0.003

See Table 3 for key.

Table 5—

Mean ± SD amino acid concentrations in serum samples obtained from 8 healthy dogs measured immediately (baseline) or after storage at −80°C for 7, 19, or 60 days with a GC-MS assay (32 runs/amino acid).

Amino acidBaselineDay 7Day 19Day 60P value
Glycine20.5 ± 5.7a19.5 ± 5.0a75.9 ± 28.1b41.9 ± 21.3a0.01
Glutamic acid3.5 ± 0.9a3.2 ± 1.0a7.0 ± 1.2b23.8 ± 3.7b0.001
Methionine5.7 ± 1.5a5.2 ± 1.2a7.2 ± 2.0b7.8 ± 2.1b0.01
Tyrosine7.2 ± 1.6a7.0 ± 1.2a4.6 ± 0.6b5.2 ± 1.8b0.01
Tryptophan182.3 ± 71.0a133.0 ± 33.8a172.0 ± 40.0a129.4 ± 32.8a0.006
Lysine31.8 ± 24.4a51.1 ± 21.4a34.3 ± 9.9b55.7 ± 19.1b0.001

See Table 3 for key.

For the first 7 days of sample storage at −20°C, serum concentrations of glycine, glutamic acid, methionine, tyrosine, and tryptophan did not change significantly. Lysine concentration increased significantly (P = 0.003) from baseline after 7 days of sample storage at −20°C. After 19 days of storage at −20°C, serum glycine and glutamic concentrations were significantly (P = 0.01) greater than at baseline, and tyrosine concentration was significantly (P = 0.01) less than at baseline. No significant changes were observed for methionine, tryptophan, and lysine concentrations in the same conditions.

No significant changes from baseline in serum amino acid concentrations were identified for any of the 6 amino acids through 7 days of sample storage at a temperature of −80°C. After 19 days of storage at −80°C, serum glycine (P = 0.01), glutamic acid (P = 0.01), and methionine (P = 0.01) concentrations were significantly greater than at baseline and tyrosine (P = 0.01) and lysine (P = 0.01) concentrations were significantly less than at baseline. Similar changes in glutamic acid (P = 0.001), methionine (P = 0.01), and lysine (P = 0.001) were identified for serum samples stored at −80°C for 60 days. On the other hand, no significant changes from baseline in serum glycine and tryptophan concentrations were identified after storage at −80°C for 60 days.

Discussion

Gas chromatography–mass spectrometry is a commonly used technique to quantify amino acids in biological samples. The first objective of the present study was to validate a GC-MS method for the measurement of glycine, glutamic acid, methionine, tyrosine, tryptophan, and lysine concentrations in pooled canine serum samples. The second objective was to evaluate the stability of these amino acids in serum samples from dogs under various storage conditions.

Findings suggested that the method used for amino acid extraction from canine serum samples in the present study was efficient, without the need of a solid-phase extraction step. Use of the technique could maximize use of time, facilitate automation of serum samples, and reduce assay variability during measurement of amino acid concentrations.31 Several variables involved in the validation of amino acid measurements were examined, including LOD, spiking recovery, intra- and interassay variability, and dilutional parallelism. An intra-assay CV < 15% was achieved, which is within the expected range for intra- and interassay validation of analytes via GC-MS,33 indicating acceptable precision and reproducibility of the method used. Interassay CVs for all amino acids were ≤ 15% (ie, acceptable), except for glycine (16.6%). In that situation, storage of serum aliquots at −80°C until analysis for determination of interassay variability appeared to lead to a decrease in glycine stability. Results obtained from the tests in which mean percentage recoveries were calculated for all 6 amino acids suggested that the procedure for derivatization by use of MSTFA was satisfactory. Spiking recoveries for all 6 amino acids indicated that this method was quantitative and precise (79.9% to 112.7% recovery of added, known amounts of amino acids). In addition, calibration curves generated for each of the 6 amino acids by use of 6 standard amino acid concentrations provided evidence for the accuracy of the method described, with mean percentage recoveries ranging from 80% to 120%. Ratios of observed to expected concentrations (77.8% to 123.0%) in dilutional parallelism tests for all 6 amino acids indicated acceptable linearity. Limits of detection for all 6 amino acids ranged from 2.6 to 6.1 μmol/L. We therefore concluded that the GC-MS method yielded linear, precise, accurate, and reproducible results for measurement of glutamic acid, glycine, methionine, tyrosine, tryptophan, and lysine concentrations in canine serum samples.

Several factors can affect amino acid concentrations in serum samples from various animal species including storage duration,34,35 storage temperature,36 sample collection technique, and hemolysis as well as proteolytic and enzymatic activity within a sample.34,37 Enzymatic activity is considered an important cause of changes in amino acid concentrations in blood samples stored at room temperature.36 Hydrolysis of proteins is catalyzed by proteases and may lead to an increase in amino acid concentrations in biological samples as a result of protein degradation and release of small peptides.35 To neutralize enzymatic activity, storage of biological samples at −80°C is warranted.34,36 Arginase activity may play a role in the decrease in arginine concentration and increase in ornithine concentration that occur when rat plasma is stored at −80°C.34 An increase in arginase activity has been detected in hemolyzed plasma and serum samples caused by rupture of RBCs and leukocytes.36 For this reason, extraction of amino acids was performed immediately after blood sample collection in the present study to assess concentrations at the point of sample collection. In addition, fresh internal standards were prepared daily to avoid degradation of the internal standards and subsequent quantification errors.

Stability analyses of serum amino acid concentrations revealed no significant changes for any amino acid when serum samples were stored for 7 days at −80°C. A significant increase in lysine concentration was evident after sample storage at 4°C for ≥ 1 day. Similarly, an increase in serum glutamic acid concentration was identified after 1 week of storage at 4°C. Moreover, a significant increase in serum glutamic acid concentration was identified after 19 days of storage at −20°C. These findings concurred with data from a study35 conducted to assess the stability of amino acids in plasma samples obtained from Holstein cows and stored for 2 weeks in the same conditions as in the present study. A previous study36 involving human plasma samples also revealed that most amino acid concentrations increase by > 10% when samples are stored at room temperature for > 24 hours. Indeed, a 5-fold increase in glutamic acid concentration was detected in human plasma samples after storage at room temperature for 24 hours,36 and a similar increase in glutamic acid concentration was also identified for stored rat plasma samples.34 In the study reported here, serum glutamic acid concentration increased from baseline by 33% after storage of serum samples for 1 week at 4°C, and an increase of > 200% was identified for the same amino acid after storage for 19 days at −20°C or −80°C. The reason for the increase in serum amino acid concentrations after serum sample storage is speculated to be hydrolysis of small proteins induced by changes in pH or temperature,37 which releases amino acids.

In contrast, no significant changes in serum methionine or tryptophan concentrations were identified in the present study after storage of samples at 4°C for 1 week or at −20°C for 19 days. Serum glycine and tryptophan concentrations did not change significantly from baseline when samples were stored for 60 days at −80°C; however, serum glutamic acid, methionine, and lysine concentrations increased significantly in the same conditions. Serum tyrosine concentration had a different pattern in that it decreased after storage for 19 days at −20°C or −80°C or for 60 days. Similar changes in tyrosine concentrations were identified in human serum samples stored for 3 months at −20°C.38 The present findings suggested that it will be important to consider amino acid stability in future clinical and research applications.

One limitation of the study reported here was that the GC-MS method was evaluated for analysis of only 6 amino acids, primarily because of the high cost of the internal standards that would be needed to quantify other amino acids reliably. Another reason was that some of the nonevaluated amino acids, including arginine, serine, and threonine, are known to degrade during the analysis process because of thermal volatility or column adherence during GC-MS.

In the present study, the validity of a GC-MS method for quantification of glutamic acid, glycine, methionine, tyrosine, tryptophan, and lysine concentrations in canine serum samples was evaluated. For each of these amino acid assays, results were linear, precise, accurate, and reproducible. Amino acid concentration measurements indicated the amino acids analyzed were stable for 1 week when serum samples were stored at −80°C. Therefore, the methods used in the present study could be useful for assessment of serum amino acid concentrations in dogs with gastrointestinal and other diseases.

Acknowledgments

Presented in part at the 2014 Annual Forum of the American College of Veterinary Internal Medicine, Nashville, Tenn, June 2014.

ABBREVIATIONS

CV

Coefficient of variation

GC-MS

Gas chromatography–mass spectrometry

LOD

Limit of detection

MSTFA

N-methyl-N-(trimethylsilyl)trifluoroacetamide

Footnotes

a.

Glycine-2,2-d2, C/D/N Isotopes Inc, Pointe-Claire, QC, Canada.

b.

DL-glutamic-2,4,4-d3 acid, C/D/N Isotopes Inc, Pointe-Claire, QC, Canada.

c.

L-tryptophan-2,4,5,6,7-d5, C/D/N Isotopes Inc, Pointe-Claire, QC, Canada.

d.

L-lysine-4,4,5,5-d4 hydrochloride, C/D/N Isotopes Inc, Pointe-Claire, QC, Canada.

e.

L-methione-methyl-d3, Cambridge Isotope Laboratories, Andover, Mass.

f.

L-tyrosine 3, 3-d2, Cambridge Isotope Laboratories, Andover, Mass.

g.

Sigma Chemical Co, St Louis, Mo.

h.

Eppendorf microcentrifuge model 5430, Eppendorf AG, Hamburg, Germany.

i.

Eppendorf Vacufuge Plus, Eppendorf AG, Hamburg, Germany.

j.

VF-17ms 50% phenyl, 50% dimethylpolysiloxane column (30 × 0.25 [0.25]), Agilent Technologies, Santa Clara, Calif.

k.

7890 model gas chromatograph, Agilent Technologies, Santa Clara, Calif.

l.

5975C mass selective detector, Agilent Technologies, Santa Clara, Calif.

m.

Vacutainer, Becton, Dickinson & Co, Franklin Lakes, NJ.

n.

GraphPad Prism, version 5.0, GraphPad Software, La Jolla, Calif.

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Contributor Notes

Address correspondence to Dr. Lopes (rlopes@cvm.tamu.edu).