Liquid Chromatography-electrospray Ionization-mass Spectrometric Research Articles

Food authentication goes green: Determination of the geographical origin of grain maize (Zea mays L.) using direct analysis in real time mass spectrometry (DART-MS)

Time savings, environmental friendliness and cost reduction are important criteria to determine the authenticity of feed and food in routine analysis. One analytical technique that is especially promising in terms of these criteria in metabolomics studies is Direct Analysis in Real Time Mass Spectrometry (DART-MS). In the present study, DART-MS is applied to 200 grain maize samples from seven geographical origins to develop a data processing workflow and an approach for origin analysis of grain maize. For the first time, such a large sample set was investigated using a DART-MS-based non-targeted lipidomics approach. Various open-source programs were used for pre-processing and random forest (RF) combined with repeated cross-validation was used for classification, resulting in an accuracy of 84.4%. Subsequently, the most important markers identified by RF, belonging to the classes of mono-, di- and triacylglycerols, were identified using DART-MS2. The chosen method significantly minimizes the time and solvent consumption compared to classic liquid chromatography electrospray ionization mass spectrometric (LC-ESI-MS) strategies and thus offers a more cost-effective and environmentally friendly alternative. Furthermore, the results show the high potential for determining the geographical origin of grain maize using DART-MS.

Rapid detection and identification of dioscorine compounds in Dioscorea hispida tuber plants by LC-ESI-MS

Studies have revealed that Dioscorea hispida tubers contain a poisonous substance called alkaloid dioscorine. The method for removing alkaloid dioscorine in Dioscorea hispida is explored in this research through a soaking process. The tubers were peeled, washed, sliced, and soaked for 5 days in either 1.0 M sodium chloride (NaCl) or distilled water. The aim of this study was to firstly identify the amount of toxic dioscorine remaining after soaking for 5 days, and then determine the best solution for removing dioscorine compounds in the tubers that were obtained from a tropical area in Peninsular Malaysia. The liquid chromatography electrospray ionization mass spectrometric (LC-ESI-MS) systems were used to identify the presence of alkaloid dioscorine compounds within Dioscorea hispida tubers. The analysis showed that no dioscorine compounds were present in day 5 for samples soaked in the NaCl solution. However, the relative abundance in the distilled water solution at day 5 was 281000, indicating a 95% decrease of the relative abundance value of the dioscorine compounds. This work aimed to determine the minimum days needed to remove the poisonous element before Dioscorea hispida tubers could be used for food consumption or for any other applications.

High performance liquid chromatography-electrospray ionization mass spectrometric (LC-ESI-MS) methodology for analysis of amino acid energy substrates in microwave-fixed microdissected brain tissue

Hypoglycemia deprives the brain of its primary energy source glucose. Reductions in whole-brain amino acid energy substrate levels suggest that these non-glucose fuels may be metabolized during glucose shortage. Recurring hypoglycemia can cause mal-adaptive impairment of glucose counter-regulation; yet, it is unclear if amplified reliance upon alternative metabolic substrates impedes detection of continuing neuro-glucopenia. This research aimed to develop high-sensitivity UHPLC-electrospray ionization mass spectrometric (LC-ESI-MS) methodology, for complementary use with high-neuroanatomical resolution microdissection tools, for measurement of glucogenic amino acid, e.g. glutamine (Gln), glutamate (Glu), and aspartate (Asp) content in the characterized glucose-sensing ventromedial hypothalamic nucleus (VMN) during acute versus chronic hypoglycemia. Results show that VMN tissue Gln, Glu, and Asp levels were significantly decreased during a single hypoglycemic episode, and that Gln and Asp measures were correspondingly normalized or further diminished during renewed hypoglycemia. Results provide proof-of-principle that LC-ESI-MS has requisite sensitivity for amino acid energy substrate quantification in distinctive brain gluco-regulatory structures under conditions of eu- versus hypoglycemia. This novel combinatory methodology will support ongoing efforts to determine how amino acid energy yield may impact VMN metabolic sensory function during persistent hypoglycemia.

Comparison of liquid chromatography-microchip/mass spectrometry to conventional liquid chromatography–mass spectrometry for the analysis of steroids

The feasibility of a microfluidic-based liquid chromatography-electrospray ionization/mass spectrometric system (HPLC-Chip/ESI/MS) was studied and compared to a conventional narrow-bore liquid chromatography-electrospray ionization/mass spectrometric (LC-ESI/MS) system for the analysis of steroids. The limits of detection (LODs) for oxime derivatized steroids, expressed as concentrations, were slightly higher with the HPLC-Chip/MS system (50–300pM) using an injection volume of 0.5μL than with the conventional LC-ESI/MS (10–150pM) using an injection volume of 40μL. However, when the LODs are expressed as injected amounts, the sensitivity of the HPLC-Chip/MS system was about 50 times higher than with the conventional LC-ESI/MS system. The results indicate that the use of HPLC-Chip/MS system is clearly advantageous only in the analysis of low-volume samples. Both methods showed good linearity and good quantitative and chromatographic repeatability. In addition to the instrument comparisons with oxime derivatized steroids, the feasibility of the HPLC-Chip/MS system in the analysis of non-derivatized and oxime derivatized steroids was compared. The HPLC-Chip/MS method developed for non-derivatized steroids was also applied to the quantitative analysis of 15 mouse plasma samples.

Study of liquid chromatography/electrospray ionization mass spectrometry matrix effect on the example of glyphosate analysis from cereals

Glyphosate is one of the most common pesticides used in the pre-harvest treatment of cereals. This paper examines the matrix effect of glyphosate liquid chromatography/electrospray ionization mass spectrometric (LC/ESI-MS) analysis in wheat and rye. The matrix effect (ionization suppression) was found to be dependent on sample particle size taken for the extraction. If samples are ground to very small particles severe ionization suppression occurs. For lower glyphosate contents (<1 mg/kg) the signal may even be suppressed by more than 90%. The matrix effect was found to be dependent on the matrix - rye showed significantly stronger ionization suppression than wheat, although these matrices are not very different. The matrix effect also depends on the concentration of glyphosate in the post-extraction spiked samples. It is demonstrated that the isotope-labelled standard (13)C(2)-glyphosate undergoes different ionization suppression than glyphosate and is therefore not efficient in compensating for matrix effect. At the same time the extrapolative dilution approach allows to efficiently compensate for matrix effect.

Development and validation of high‐performance liquid chromatography/electrospray ionization mass spectrometry for assay of madecassoside in rat plasma and its application to pharmacokinetic study

A rapid, sensitive and specific high-performance liquid chromatography/electrospray ionization mass spectrometric (LC-ESI-MS) method was developed and validated for the quantification of madecassoside, a major active constituent of Centella asiatica (L.) Urb. herbs, in rat plasma. With paeoniflorin as an internal standard (IS), a simple liquid-liquid extraction process was employed for the plasma sample preparation. Chromatographic separation was achieved within 6 min on a Shim-pack CLC-ODS column using acetonitrile and water (60:40, v/v) containing 0.1% (v/v) formic acid as the mobile phase. The detection was performed by MS with electrospray ionization interface in negative selected ion monitoring (SIM) mode. The linear range was 11-5500 ng/mL with the square regression coefficient (r(2) ) of 0.9995. The lower limit of quantification was 11 ng/mL. The intra- and inter- day precision ranged from 4.99 to 9.03%, and the accuracy was between 95.82 and 111.80%. The average recoveries of madecassoside and IS from spiked plasma samples were >92%. The developed method was successfully applied to the pharmacokinetic study of madecassoside in rats after an oral administration.

Rapid determination of rifaximin on dried blood spots by LC‐ESI‐MS

The use of blood spot collection cards is a simple way to obtain specimens for therapeutic drug monitoring, assessing adherence to medications and preventing toxicity in a clinical setting. A high-throughput liquid chromatography-electrospray ionization mass spectrometric (LC-ESI-MS) method for determination of rifaximin on dried blood spots (DBS) was developed and validated. It involves solvent extraction of a punch of DBS followed by reversed-phase LC on a monolithic column consisting of a silica rod with bimodal pore structure and detection by ESI-MS. Rifampicin was used as an internal standard (IS). The run time was within 5.0 min with a very low back-pressure at a flow rate of 0.5 mL/min. The assay was linear from 0.1 to 10 ng/mL. The mean recovery was 98.42%. The developed method is very simple, rapid and useful for clinical applications.

Development of a validated high‐throughput LC‐ESI‐MS method for determination of sirolimus on dried blood spots

A high-throughput liquid chromatography-electrospray ionization mass spectrometric (LC-ESI-MS) method for screening of sirolimus on dried blood spots (DBS) was developed and validated. It involves solvent extraction of a punch of DBS followed by reversed-phase LC on a relatively new monolithic column consisting of a silica rod with bimodal pore structure and detection by ESI-MS. The run time was less than 3 min with a very low backpressure at a flow rate of 0.5 mL/min. The method can analyze more than 100 samples in an 8 h working day, including sample preparation. The assay was linear from 1 to 100 ng/mL. The mean recovery was 92.42%. The mean inter-day and intra-day precisions were 1.23 and 1.41%, respectively. The developed method is simple, rapid and useful for clinical applications.

Determination of Nimodipine in Human Plasma by HPLC-ESI-MS and Its Application to a Bioequivalence Study

A simple, specific, and precise liquid chromatographic-electrospray ionization mass spectrometric (LC-ESI-MS) method has been developed for determination of nimodipine concentration in human plasma. The method involves the addition of 200 microL of saturated sodium bicarbonate (NaHCO(3)) solution to plasma to improve the extraction recovery, liquid-liquid extraction of nimodipine from plasma samples with anhydrous diethyl ether, simple reversed-phase chromatography, and ESI mass spectrometric detection in negative ion selected ion monitoring mode (SIM) using target [M-] ions at m/z 417 and m/z 359 for nimodipine and nitrendipine (internal standard, IS), respectively. A complete analytical run was achieved within 3.5 min. The limit of quantification was 0.5 ng/mL. The method was validated within a linear range of 0.5-100 ng/mL. The correlation coefficient for the calibration regression line was 0.9995 or better. Intra- and inter-batch accuracy and precision were acceptable. Analyte was stable in a battery of stability studies. The method has been successfully used in a bioequivalence study.

LC‐MS determination and pharmacokinetic studies of paeonol in rat plasma after administration of different compatibility of Su‐Xiao‐Xin‐Tong prescriptions

A rapid and specific liquid chromatographic/electrospray ionization mass spectrometric (LC/ESI-MS) method has been developed and validated for the identification and quantification of paeonol in rat plasma. Paeonol and internal standard were isolated from plasma samples by liquid-liquid extraction with chloroform. The chromatographic separation was accomplished on a Zorbax-SB C18 column (100x2.1 mm, 3.5 microm). The mobile phase consisted of acetonitrile and 0.1% aqueous formic acid (64:36) was delivered at a flow rate of 0.2 mL/min. Detection was performed on a single quadrupole mass spectrometer by selected ion monitoring mode via electrospray ionization source. Linearity was established for the range of concentrations 0.0525-15.8 microg/mL with a coefficient correlation (r) of 0.9995. The intra- and inter-day precision (RSD%) was lower than 9.34% and accuracy ranged from 93.7 to 102.3%. The lower limit of quantification was 0.0525 microg/mL. The proposed method was used to determine the concentration of paeonol for pharmacokinetic studies. The pharmacokinetics of different compatibility prescriptions of Su-Xiao-Xin-Tong were studied and compared.

Simultaneous quantification of oxysophocarpine and its active metabolite sophocarpine in rat plasma by liquid chromatography/mass spectrometry for a pharmacokinetic study

A sensitive, rapid and specific method for the simultaneous quantification of oxysophocarpine (OSC) and its active metabolite sophocarpine (SC) in rat plasma was developed and validated, using a liquid-liquid extraction procedure followed by liquid chromatography/electrospray ionization mass spectrometric (LC/ESI-MS) analysis. The separation was performed on a Zorbax Extend-C(18) column (2.1 mm i.d. x 50 mm, 5 microm) with a C(18) guard column using methanol-water containing 5 mm ammonium acetate (15:85, v/v) as mobile phase. Analysis was performed in selected ion monitoring (SIM) mode with an electrospray ionization (ESI) interface. [M + H](+) at m/z 263 for OSC, [M + H](+) at m/z 247 for SC and [M + H](+) at m/z 249 for matrine (internal standard) were selected as detecting ions, respectively. The method was linear in the concentration ranges 10-1000 ng/mL for OSC and 5-500 ng/mL for SC. The intra- and inter-day precisions (coefficient of variation) were within 7% for both analytes. Their accuracy (relative error) ranged from -6.4 to 1.5%. The limits of detection for OSC and SC were 3 and 1.5 ng/mL, respectively. The limits of quantitation for OSC and SC were 10 and 5 ng/mL, respectively. Recoveries of both analytes were greater than 85% at the low, medium and high concentrations. Both analytes were stable during all sample storage, preparation and analytic procedures. The method was successfully applied to a pharmacokinetic study after an oral administration of OSC to rats with a dose of 15 mg/kg.

Liquid Chromatography‐Electrospray Ionization Mass Spectrometric Method for Determination of Gliclazide in Human Plasma

A rapid, sensitive, and specific liquid chromatography‐electrospray ionization mass spectrometric (LC‐ESI‐MS) method has been developed for quantification of gliclazide in human plasma. The analyte and tolbutamide (internal standard, I.S.) were extracted from plasma samples with n‐hexane–dichloromethane (1:1, v/v) and analyzed on a C18 column. The chromatographic separation was achieved within 4.0 min by using methanol–0.5% formic acid (80:20, v/v) as mobile phase and the flow rate was 1.0 mL/min. Ion signals m/z 324.0 and 271.0 for gliclazide and internal standard were measured in the positive mode, respectively. The method was linear within the range of 2.5–2000 ng/mL. The lower limit of quantification (LLOQ) was 2.5 ng/mL. The intra‐ and inter‐day precisions were lower than 2.8% in terms of relative standard deviation (RSD). The inter‐day relative error (RE) as determined from quality control samples (QCs) ranged from −1.93% to 1.85%. This validated method was successfully applied for the evaluation of pharmacokinetic profiles of gliclazide modified‐release tablets in 20 healthy volunteers.

Profiling of sulfoconjugates in urine by using precursor ion and neutral loss scans in tandem mass spectrometry. Application to the investigation of heavy metal toxicity in rats.

This paper reports a liquid chromatographic/electrospray ionization mass spectrometric (LC/ESI-MS) method for profiling a wide range of structurally different sulfoconjugated compounds in urine and its application to the characterization of biomarkers for heavy metal toxicity in rat urine. Sulfoconjugates were first isolated by solid-phase extraction and the LC separation was performed on a reversed-phase column. Sulfoconjugates were detected in a triple-quadrupole mass spectrometer by simultaneously monitoring constant losses of 80 u (or 80 Th for doubly charged ions), precursors of m/z 80 (SO(3) (-*)) and precursors of m/z 97 (HSO4-). The ESI-MS detection conditions were optimized on dehydroepiandrosterone sulfate and estradiol sulfate and tested on other sulfoconjugates. The analysis of urine samples from humans and rats by using the developed method allowed the detection of about 15 peaks in each mode of detection. It was then applied to the investigation of heavy metal toxicity in rats. Comparative analysis of the chromatographic fingerprints of urine from control and uranium- and cadmium-treated rats showed several variations in the chromatographic pattern of the sulfoconjugates. Diagnostic m/z ratios were confirmed by analyzing individual urine samples and one of the observed variations seemed to be specific to uranium toxicity. The ion responsible for this variation has been identified as 4-ethylphenol sulfate by comparison of its chromatographic retention time and collision-induced dissociation mass spectra (MS(2) and MS(3) performed on a quadrupole ion trap instrument) with those of the synthesized compound.

ＬＣ−ＥＳＩ／ＭＳによる法化学的薬物・毒物分析

This paper presents a liquid chromatography-electrospray ionization-mass spectrometric (LC-MS) screening and identification procedures for drugs and toxic substances from forensic biological materials such as postmortem blood, urine, and tissues (so-called general unknown screening or systematic toxicological analysis). Gas chromatoraphy-mass spectrometry (GC-MS) has been the method of choice for toxicological analysis of volatile substances while non-volatile and/or thermally labile substances need LC-MS for identification. To develop the LC-MS procedures for forensic analysis, mass spectra library and retention indices, like in the case of GC-GM, are required. Retention indices were determined according to the retention times on the chromatographic separation using a Waters Symmetry C18 (particle size 3.5 μm), 2.1 mm × 150 mm column with a gradient of methanol and 10 mM ammonium acetate (pH 6.8) delivered at a 0.2 mL/min flow rate. Retention times of substances were then converted to retention indices based on those of uracil, phenol, methylparaben (methyl-p-hydroxybenzoate), ethylparaben (ethyl-p-hydroxybenzoate), propylparaben (propyl-p-hydroxybenzoate), butylparaben (butyl-p-hydroxybenzoate), and heptylparaben (heptyl-p-hydroxybenzoate) in the retention index standard mixture. Mass spectra library was made applying two different cone voltages (20 V and 60 V) both in the positive and negative ion modes for in-source fragmentation due to collision-induced dissociation. Mass spectra obtained presented the molecular-related ion and number of specific fragment ions sufficient for identification. The combination of the retention index, molecular-related ion and fragment ions has made LC-MS a preferred method for general unknown screening in the field of forensic toxicology to GC-MS.

Determination of methamphetamine and its metabolites incorporated in hair by column-switching liquid chromatography-mass spectrometry.

An automated column-switching liquid chromatographic-electrospray ionization-mass spectrometric (LC-ESI-MS) method has been established for the determination of methamphetamine (MA) and its metabolites, amphetamine (AP), and p-hydroxymethamphetamine (p-OH-MA) in hair. The combination of an N-vinylacetamide-containing hydrophilic polymer online extraction column, an SCX semi-micro LC column, and an electrospray ionization interface provided the successful concentration, separations, and highly sensitive MS determinations of these analytes in a hair extract without tedious sample pretreatments. The limits of detection of these analytes were 0.02 ng/mg and 0.1-0.2 ng/mg in the selected-ion monitoring (SIM) and full-scan modes, respectively, when using a 100-microL hair extract sample that corresponds to a 2.5-mg sample of hair. The calibration curves using dibenzylamine as an internal standard were linear up to 30 ng/mg hair equivalents for all these analytes in the SIM mode. p-OH-MA, the detection of which in MA users' hair had not been previously reported, was detectable in all 22 hair specimens/sections from which 1 ng or more of MA was detected per milligram hair. The amount of p-OH-MA detected per milligram of hair is presented with those of MA and AP among the MA users population. The detection of AP and p-OH-MA, in addition to the parent drug MA with reasonable ratios, was found to be a useful indicator for distinguishing internal MA incorporation from external contamination.

Gradient reversed-phase liquid chromatographic-electrospray ionization mass spectrometric method for the comparison of smokeless powders

A gradient reversed-phase liquid chromatographic-electrospray ionization mass spectrometric (LC-ESIMS) method was developed to determine compositional variation in the organic additives of smokeless powders. The method was optimized for the separation and detection of selected powder constituents, including diphenylamine, along with isomers of its nitroso and nitro derivatives, centralite I and II, in addition to dialkylphthalate acid esters. A series of commercially available smokeless powders was prepared by organic liquid extraction and characterized using the LC-ESIMS method. The results demonstrate the differentiation of smokeless powders by their additive profile.

Liquid chromatographic–mass spectrometric determination of cyclooxygenase metabolites of arachidonic acid in cultured cells

A liquid chromatographic–electrospray ionization-mass spectrometric (LC-ESI-MS) technique was developed to simultaneously determine the cyclooxygenase metabolites of arachidonic acid (6-keto-PGF 1α, PGD 2, PGE 2, PGF 2α, and PGJ 2) produced by cultured cells. Samples were separated on a C 18 column with water–acetonitrile mobile phase, ionized by electrospray, and detected in the positive mode. Selected ion monitoring (SIM) of m/ z 353, 335, 335, 319, and 317 were used for quantifying 6-keto-PGF 1α, PGD 2, PGE 2, PGF 2α, and PGJ 2, respectively. Prostaglandins were detected at concentrations as low as 1 pg ( S/ N=3) on the column. The method was used to determine the production of PGs from bovine coronary artery endothelial cells (ECs) and human prostate cancer cells (PC-3) with different degree of invasiveness. Bradykinin (10 −6 M) stimulated a marked increase in the production of 6-keto-PGF 1α, PGE 2, and PGF 2α and a small increase of PGD 2 by ECs. 6-Keto-PGF 1α was the major metabolite in these cells. The production of PGE 2 was threefold higher and PGD 2 was twofold higher in PC-3-S (invasive) cells than in PC-3-U (non-invasive) cells.

Quantitative Analysis of the DNA Adduct N2,3- Ethenoguanine Using Liquid Chromatography Electrospray Ionization Mass Spectrometry

The need for specificity and sensitivity in the analysis of DNA adducts has led the development of GC/MS methods. Such methods require chemical derivatization (i.e. silylation, electrophore labelling), which can also bring its own sets of problems, including the production of artifacts, interferences and sample to sample variability in derivatization. To obviate such problems, a liquid chromatographic/electrospray ionization mass spectrometric (LC/ESI-MS) method was developed to quantify N2,3-ethenoguanine (εGua), a promutagenic DNA adduct of vinyl chloride exposure. The response of εGua to isotopically labelled internal standard [13C4]εGua was linear (r2 = 0.999) and reproducible from 0.027 to 0.538 pmol μl-1. We obtained an accuracy of 86±14% by analyzing chloroethylene oxide (CEO)-treated calf thymus DNA enriched with authentic εGua. The analysis of CEO-treated calf thymus DNA samples not enriched with authentic εGua provided a precision of 15%. The detection limits with a signal-to-noise ratio (S/N) 2.5:1 were obtained in the determination of authentic εGua at 5 fmol per injection. The detection limit obtained in the routine analysis of the biological samples was 50 fmol εGua with S/N = 3:1. The applicability of the method was established by determining εGua in rats treated with CEO by portal vein injection and an unexposed human liver. It was observed that the concentration of εGua in the rat livers increased with increase in dose and was inversely related to the time after, CEO exposure. This trend suggests rapid repair of the adduct in rat livers. In the human liver DNA sample, εGua was quantitated at 0.06±0.01 pmol mg-1 DNA.

Quantitative analysis of the DNA adduct N2,3-ethenoguanine using liquid chromatography/electrospray ionization mass spectrometry.

The need for specificity and sensitivity in the analysis of DNA adducts has led the development of GC/MS methods. Such methods require chemical derivatization (i.e. silylation, electrophore labelling), which can also bring its own sets of problems, including the production of artifacts, interferences and sample to sample variability in derivatization. To obviate such problems, a liquid chromatographic/electrospray ionization mass spectrometric (LC/ESI-MS) method was developed to quantify N2,3-ethenoguanine (epsilon Gua), a promutagenic DNA adduct of vinyl chloride exposure. The response of epsilon Gua to isotopically labelled internal standard [13C4]epsilon Gua was linear (r2 = 0.999) and reproducible from 0.027 to 0.538 pmol microliter-1. We obtained an accuracy of 86 +/- 14% by analyzing chloroethylene oxide (CEO)-treated calf thymus DNA enriched with authentic epsilon Gua. The analysis of CEO-treated calf thymus DNA samples not enriched with authentic epsilon Gua provided a precision of 15%. The detection limits with a signal-to-noise ratio (S/N) 2.5:1 were obtained in the determination of authentic epsilon Gua at 5 fmol per injection. The detection limit obtained in the routine analysis of the biological samples was 50 fmol epsilon Gua with S/N = 3:1. The applicability of the method was established by determining epsilon Gua in rats treated with CEO by portal vein injection and an unexposed human liver. It was observed that the concentration of epsilon Gua in the rat livers increased with increase in dose and was inversely related to the time after, CEO exposure. This trend suggests rapid repair of the adduct in rat livers. In the human liver DNA sample, epsilon Gua was quantitated at 0.06 +/- 0.01 pmol mg-1 DNA.

Peptide chiral purity determination: hydrolysis in deuterated acid, derivatization with Marfey's reagent and analysis using high-performance liquid chromatography-electrospray ionization-mass spectrometry

A high-performance liquid chromatography-electrospray ionization-mass spectrometric (LC-ESI-MS) method is presented that allows rapid and accurate determination of amino acid chiral purity in a peptide. Peptides are hydrolyzed in hydrochloric acid-d 1/acetic acid-d 4 and then converted to diastereomers by derivatization with 1-fluoro-2,4-dinitrophenyl-5- l-alanine amide (FDAA, Marfey's reagent). Mixtures of d- and l-amino acid diastereomeric pairs are resolved in one chromatographic separation using conventional reversed-phase high-performance liquid chromatography. Hydrolysis in a deuterated solvent is necessary because the original ratio of d-/l-amino acids present in a peptide changes during acid hydrolysis due to racemization. Peptide hydrolysis in deuterated acids circumvents this problem by labeling each amino acid that racemizes with one deuterium at the α-carbon. An increase in molecular mass of one atomic mass unit allows racemized amino acids to be distinguished from non-racemized amino acids by mass spectrometry. This procedure was used to determine the chiral purity of each amino acid in a purified, hexapeptide by-product (Arg-Lys-Lys-Asp-Val-Tyr) present in a kilogram batch of the synthetic pentapeptide, thymopentin (Arg-Lys-Asp-Val-Tyr).