Analysis of cannabinoids by liquid chromatography— Thermospray mass spectrometry and liquid chromatography— Tandem mass spectrometry

Summary A rapid method based on liquid chromatography and thermospray mass spectrometry without any derivatization or pre-purification steps has been developed for the identification and quantification of cannabinoids in drugs from cannabis plants. The extracts were separated on a C₁₈ reversed-phase column with an acidic acetonitrile-water gradient. Liquid chromatographymass spectrometry was performed with a thermospray interface and protonated molecular ions were obtained from the cannabinoids of interest. Liquid chromatography-tandem mass spectrometry experiments on the molecular ions gave additional structural information online. The sensitivity and selectivity of the method was sufficient to enable the detection of 100 pg of the cannabinoids.     

Analysis of pharmaceutical impurities using multi‐heartcutting 2D LC coupled with UV‐charged aerosol MS detection

To overcome challenges in HPLC impurity analysis of pharmaceuticals, we developed an automated online multi‐heartcutting 2D HPLC system with hyphenated UV‐charged aerosol MS detection. The first dimension has a primary column and the second dimension has six orthogonal columns to enhance flexibility and selectivity. The two dimensions were interfaced by a pair of switching valves equipped with six trapping loops that allow multi‐heartcutting of peaks of interest in the first dimension and also allow “peak parking.” The hyphenated UV‐charged aerosol MS detection provides comprehensive detection for compounds with and without UV chromophores, organics, and inorganics. It also provides structural information for impurity identification. A hidden degradation product that co‐eluted with the drug main peak was revealed by RP × RP separation and thus enabled the stability‐indicating method development. A poorly retained polar component with no UV chromophores was analyzed by RP × hydrophilic interaction liquid chromatography separation with charged aerosol detection. Furthermore, using this system, the structures of low‐level impurities separated by a method using nonvolatile phosphate buffer were identified and tracked by MS in the second dimension.     

Analysis of 30 synthetic cannabinoids in serum by liquid chromatography‐electrospray ionization tandem mass spectrometry after liquid‐liquid extraction

The analysis of synthetic cannabinoids in human matrices is of particular importance in the fields of forensic and clinical toxicology since cannabis users partly shift to the consumption of ‘herbal mixtures’ as a legal alternative to cannabis products in order to circumvent drug testing. However, comprehensive methods covering the majority of synthetic cannabinoids already identified on the drug market are still lacking. In this article, we present a fully validated method for the analysis of 30 synthetic cannabinoids in human serum utilizing liquid‐liquid extraction and liquid chromatography‐electrospray ionization tandem mass spectrometry. The method proved to be suitable for the quantification of 27 substances. The limits of detection ranged from 0.01 to 2.0 ng/mL, whereas the lower limits of quantification were in the range from 0.1 to 2.0 ng/mL. The presented method was successfully applied to 833 authentic serum samples during routine analysis between August 2011 and January 2012. A total of 227 (27%) samples was tested positive for at least one of the following synthetic cannabinoids: JWH‐018, JWH‐019, JWH‐073, JWH‐081, JWH‐122, JWH‐200, JWH‐203, JWH‐210, JWH‐307, AM‐2201 and RCS‐4. The most prevalent compounds in positive samples were JWH‐210 (80%), JWH‐122 (63%) as well as AM‐2201 (29%). Median serum concentrations were all below 1.0 ng/mL. These findings demonstrate a significant shift of the market of synthetic cannabinoids towards substances featuring a higher CB₁ binding affinity and clearly emphasize that the analysis of synthetic cannabinoids in serum or blood samples requires highly sensitive analytical methods covering a wide spectrum of substances. Copyright © 2012 John Wiley & Sons, Ltd.     

Qualitative and quantitative analysis of THC, 11‐hydroxy‐THC and 11‐nor‐9‐carboxy‐THC in whole blood by ultra‐performance liquid chromatography/tandem mass spectrometry

A qualitative and quantitative analytical method was developed for the simultaneous determination of Δ⁹‐tetrahydrocannabinol (THC), 11‐hydroxy‐Δ⁹‐tetrahydrocannabinol (11‐OH‐THC) and l1‐nor‐9‐carboxy‐Δ⁹‐tetrahydrocannabinol (THC‐COOH) in whole blood. The samples were prepared by solid‐phase extraction followed by ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) analysis using positive ion electrospray ionization and multiple reaction monitoring. The chromatographic separation was performed with an Acquity UPLC® HSS T3 (50 × 2.1 mm i.d., 1.8 µm) reversed‐phase column using a methanol/2 mM ammonium formate (formic acid 0.1%) gradient in a total run time of 9.5 min. MS/MS detection was achieved with two precursor‐product ion transitions per substance. The method was fully validated, including selectivity and capacity of identification, according to the identification criteria (two transitions per substance, signal‐to‐noise ratio, relative retention time and ion ratio) without the presence of interferences, limit of detection (0.2 µg/L for THC and 0.5 µg/L for 11‐OH‐THC and THC‐COOH), limit of quantitation (0.5 µg/L for all cannabinoids), recovery (53–115%), carryover, matrix effect (34‐43%), linearity (0.5‐100 µg/L), intra‐assay precision (CV < 10% for the relative peak area ratios and <0.1% for the relative retention time), inter‐assay accuracy (mean relative error <10%) and precision (CV <11%). The method has already been successfully used in proficiency tests and subsequently applied to authentic samples in routine forensic analysis. Copyright © 2011 John Wiley & Sons, Ltd.     

Application of optically pure amines as chiral auxiliaries to develop trichloro‐s‐triazine‐based new chiral derivatizing reagents for reversed‐phase high‐performance liquid chromatographic enantioseparation of dl‐selenomethionine

(R)‐(+)‐naphthylethyl amine and (S)‐(+)‐1‐benzyl‐3‐aminopyrrolidine were incorporated as chiral auxiliaries, by nucleophilic substitution of chlorine atoms, in cyanuric chloride (CC) or its 6‐butoxy derivative. There were obtained four new chiral derivatizing reagents (CDRs) as two dichloro and two monochloro triazine reagents. The CDRs so obtained were characterized and their optical purity was ascertained. Diastereomers of dl ‐selenomethionine were synthesized under microwave irradiation for 60 or 90 s (at 80% power of 800 W). Reversed‐phase high‐performance liquid chromatographic separation of diastereomers was carried out on a C₁₈ column using mixtures of acetonitrile with aqueous trifluoroacetic acid as mobile phase. The detection was made at 230 nm using a photodiode array detector. The separation behaviors in terms of retention times and resolutions were compared. The separation method was validated for limit of detection, linearity, accuracy, precision, and recovery. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of Doxepin in human plasma using cloud‐point extraction with high‐performance liquid chromatography and ultraviolet detection

A new straightforward method based on cloud‐point extraction has been developed, optimized, and validated for the determination of doxepin in human plasma by high‐performance liquid chromatography separation and UV detection. The nonionic surfactant Triton X‐114 was chosen as the extraction solvent. Chromatography separation was performed on a μBondapak^R C₁₈ column (4.6 mm id × 300 mm, 3 μm particle size), which was used for isocratic elution at a detection wavelength of 289 nm. Under the optimum conditions, the linear range of doxepin in human plasma was 0.1–0.9 μg/mL. Also, the detection limit, preconcentration factor, and enrichment factor were 0.08 μg/mL, 50, and 49.0, respectively.     

Quadrupole‐time‐of‐flight mass spectrometry screening for synthetic cannabinoids in herbal blends

‘Legal highs’ are novel substances which are intended to elicit a psychoactive response. They are sold from ‘head shops’, the internet and from street suppliers and may be possessed without legal restriction. Several months ago, a 19‐year‐old woman came searching for medical treatment as she had health problems caused by smoking legal highs. The substances were sold as herbal blends in plastic bags under four different labels. In this work, samples of these herbal blends have been analysed to investigate the presence of psychoactive substances without any reference standard being available at the laboratory. A screening strategy for a large number of synthetic and natural cannabinoids has been applied based on the use of ultra‐high pressure liquid chromatography coupled to quadrupole‐time of flight mass spectrometry (UHPLC‐QTOF MS) under MS^E mode. A customized home‐made database containing literature‐based exact masses for parent and product ions of around 200 synthetic and natural cannabinoids was compiled. The presence of the (de)protonated molecule measured at its accurate mass was evaluated in the samples. When a peak was detected, collision‐induced dissociation fragments and characteristic isotopic ions were also evaluated and used for tentative identification. After this tentative identification, four synthetic cannabinoids (JWH‐081, JWH‐250, JWH‐203 and JWH‐019) were unequivocally confirmed by subsequent acquisition of reference standards. The presence in the herbal blends of these synthetic cannabinoids might explain the psychotic and catatonic symptoms observed in the patient, as JWH compounds could act as potent agonists of CB₁ and CB₂ receptors located in the Limbic System and Basal ganglia of the human brain. Copyright © 2013 John Wiley & Sons, Ltd.     

Hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry method for the simultaneous determination of l‐valine,l‐leucine,l‐isoleucine,l‐phenylalanine,and l‐tyrosine in human serum

l ‐Valine, l ‐leucine, l ‐isoleucine, l ‐phenylalanine, and l ‐tyrosine are important proposed biomarkers for the early detection and diagnosis of type 2 diabetes. A simple and selective hydrophilic interaction chromatography with tandem mass spectrometry method was developed for the simultaneous determination of these amino acids in human serum, using stable isotope‐labeled amino acids as internal standards. Chromatographic separation was carried out on a Syncronis HILIC column (150 mm × 2.1 mm, 5 μm) with the column temperature of 35°C and a mobile phase consisted of acetonitrile/120 mM ammonium acetate (89:11, v/v), and the run time was 11.0 min. The mass spectrometric analysis was performed using a QTRAP 5500 mass spectrometer coupled with an electrospray ionization source in positive ion mode. As these five amino acids are endogenous compounds in serum, we used the corresponding stable isotope‐labeled amino acids to evaluate the matrix effect and recovery in serum. The matrix effect was 98.7–107.3%, and the recovery was 92.7–102.3%. Calibration curves spiked unlabeled amino acids in water were linear over the range of 0.200–100 μg/mL. The accuracy, inter‐, and intraday precision were below 10.2%. Analytes were stable during the study. This assay method has been validated and applied to the early diagnosis research of type 2 diabetes.     

Chromatographic behavior of small organic compounds in low‐temperature high‐performance liquid chromatography using liquid carbon dioxide as the mobile phase

Low‐temperature high‐performance liquid chromatography, in which a loop injector, column, and detection cell were refrigerated at –35ºC, using liquid carbon dioxide as the mobile phase was developed. Small organic compounds (polyaromatic hydrocarbons, alkylbenzenes, and quinones) were separated by low‐temperature high‐performance liquid chromatography at temperatures from –35 to –5ºC. The combination of liquid carbon dioxide mobile phase with an octadecyl‐silica (C₁₈) column provided reversed phase mode separation, and a bare silica‐gel column resulted in normal phase mode separation. In both the cases, nonlinear behavior at approximately –15ºC was found in the relationship between the temperature and the retention factors of the analytes (van't Hoff plots). In contrast to general trends in high‐performance liquid chromatography, the decrease in temperature enhanced the separation efficiency of both the columns.     

Stereoisomer separation of flavanones and flavanone‐7‐O‐glycosides by means of nanoliquid chromatography employing derivatized β‐cyclodextrins as mobile‐phase additive

A nanoliquid chromatographic method for the stereoisomer separation of some flavanone aglycones and 7‐O‐glycosides has been proposed employing a C18 capillary column and a chiral mobile‐phase additive such as cyclodextrin. The chiral separation of eriodictyol, naringenin, and hesperitin was obtained by addition of carboxymethyl‐β‐cyclodextrin to the mobile phase, whereas eriocitrin, naringin, narirutin, and hesperidin diastereoisomers were resolved by using sulfobutyl ether‐β‐cyclodextrin. The influence of the composition of the mobile phase, the length of the capillary column, and the flow rate on the chiral recognition were investigated. At optimum conditions, baseline separation for the selected aglycones and glycosylated forms were achieved with a mobile phase consisting of 50 mM sodium acetate buffer pH 3 and 30% methanol containing 20 mM of carboxymethyl‐β‐cyclodextrin and 10 mM of sulfobutyl ether‐β‐cyclodextrin, respectively. Precision, linearity, and sensitivity of the method were tested. Limits of detection and quantification for the studied flavanone glycosides were in the range 1.3‐2.5 and 7.5‐12.5 µg/mL, respectively. The method was used for the determination of the diastereomeric composition of the flavanone‐7‐O‐glycosides in Citrus juices after solid‐phase extraction procedure.     

Ultraperformance convergence chromatography‐high resolution tandem mass spectrometry for lipid biomarker profiling and identification

Lipids represent biologically ubiquitous and highly dynamic molecules in terms of abundance and structural diversity. Whereas the potential for lipids to inform on disease/injury is promising, their unique characteristics make detection and identification of lipids from biological samples analytically demanding. We report the use of ultraperformance convergence chromatography (UPC²), a variant of supercritical fluid chromatography, coupled to high‐resolution, data‐independent tandem mass spectrometry for characterization of total lipid extracts from mouse lung tissue. The UPC² platform resulted in lipid class separation and when combined with orthogonal column chemistries yielded chromatographic separation of intra‐class species based on acyl chain hydrophobicity. Moreover, the combined approach of using UPC² with orthogonal column chemistries, accurate mass measurements, time‐aligned low‐ and high‐collision energy total ion chromatograms, and positive and negative ion mode product ion spectra correlation allowed for confident lipid identification. Of great interest was the identification of differentially expressed ceramides that were elevated 24 h post whole thorax lung irradiation. The identification of lipids that were elevated 24 h post‐irradiation signifies a unique opportunity to investigate early mechanisms of action prior to the onset of clinical symptoms in the whole thorax lung irradiation mouse model.     

Combination of liquid‐based column separations with surface‐enhanced Raman spectroscopy

Surface‐enhanced Raman spectroscopy is a constantly developing analytical method providing not only high‐sensitive quantitative but also qualitative information on an analyte. Thus, it is reasonable that it has been tested as a promising detection method in column separations. Although its implementation in analytical separations is not widespread, some surprising results, like enormous signal enhancement and demonstrations of single‐molecule identifications, proved in only a few special examples, indicate the potential of the method. The high detection sensitivity and selectivity would be of paramount importance in trace analyses of biologically relevant molecules in complex matrices. However, the combination of surface‐enhanced Raman spectroscopy with column separation methods brings two principal issues. Interactions of analytes with metal substrates can cause deteriorations of separations and the detection can be affected by background electrolytes or elution agents. Thus, in principle, this review is on the experimental and methodological solutions to these problems. First, theoretical and practical aspects of Raman scattering, and excitation of surface plasmon in colloid suspensions of nanoparticles and on planar nanostructured substrates are briefly explained. Advances in experimental arrangements of on‐line and at‐line couplings with column liquid phase separation methods, including microfluidic devices, are described together with chosen analytical applications.     

Development of Gas-Chromatographic Method for Simultaneous Determination of Cannabinoids and Terpenes in Hemp

An original gas chromatographic method has been developed for simultaneous determination of major terpenes and cannabinoids in plant samples and their extracts. The main issues to be addressed were the large differences in polarity and volatility between both groups of analytes, but also the need for an exhaustive decarboxylation of cannabinoid acidic forms. Sample preparation was minimised, also by avoiding any analyte derivatisation. Acetone was found to be the most appropriate extraction solvent. Successful chromatographic separation was achieved by using a medium polarity column. Limits of detection ranged from 120 to 260 ng/mL for terpenes and from 660 to 860 ng/mL for cannabinoids. Parallel testing proved the results for cannabinoids are comparable to those obtained from established HPLC methods. Despite very large differences in concentrations between both analyte groups, a linear range between 1 and 100 µg/mL for terpenes and between 10 and 1500 µg/mL for cannabinoids was determined.     

A semi‐automated solid‐phase extraction liquid chromatography/tandem mass spectrometry method for the analysis of tetrahydrocannabinol and metabolites in whole blood

Marijuana is one of the most commonly abused illicit substances in the USA, making cannabinoids important to detect in clinical and forensic toxicology laboratories. Historically, cannabinoids in biological fluids have been derivatized and analyzed by gas chromatography/mass spectrometry (GC/MS). There has been a gradual shift in many laboratories towards liquid chromatography/mass spectrometry (LC/MS) for this analysis due to its improved sensitivity and reduced sample preparation compared with GC/MS procedures. This paper reports a validated method for the analysis of Δ⁹‐tetrahydrocannabinol (THC) and its two main metabolites, 11‐nor‐9‐carboxy‐Δ⁹‐tetrahydrocannabinol (THC‐COOH) and 11‐hydroxy‐Δ⁹‐tetrahydrocannabinol (THC‐OH), in whole blood samples. The method has also been validated for cannabinol (CBD) and cannabidiol (CDN), two cannabinoids that were shown not to interfere with the method. This method has been successfully applied to samples both from living people and from deceased individuals obtained during autopsy. This method utilizes online solid‐phase extraction (SPE) with LC/MS. Pretreatment of samples involves protein precipitation, sample concentration, ultracentrifugation, and reconstitution. The online SPE procedure was developed using Hysphere C8‐EC sorbent. A chromatographic gradient with an Xterra MS C₁₈ column was used for the separation. Four multiple‐reaction monitoring (MRM) transitions were monitored for each analyte and internal standard. Linearity generally fell between 2 and 200 ng/mL. The limits of detection (LODs) ranged from 0.5 to 3 ng/mL and the limits of quantitation (LOQs) ranged from 2 to 8 ng/mL. The bias and imprecision were determined using a simple analysis of variance (ANOVA: single factor). The results demonstrate bias as <7%, and imprecision as <9%, for all components at each quantity control level. Published in 2009 by John Wiley & Sons, Ltd.     

Application of 10‐ethyl‐acridine‐3‐sulfonyl chloride for HPLC determination of aliphatic amines in environmental water using fluorescence and APCI‐MS

A simple, sensitive method for the determination of aliphatic amines based on a sulfonylation reaction using 10‐ethyl‐acridine‐3‐sulfonyl chloride (EASC) as pre‐column labeling reagent with fluorescence detection and APCI‐MS identification has been developed. The labeled derivatives exhibited high stability and were enough to be efficiently analyzed by HPLC with an excitation maximum at λ_ex 270 nm and an emission maximum at λ_em 430 nm. Identification of derivatives was carried out by online post‐column MS in positive‐ion mode. Comparing with the widely used 5‐dimethylaminonaphthalene‐1‐sulfonylchloride (Dansyl‐Cl), EASC‐amine derivatives not only exhibited high fluorescence but also exhibited excellent MS ionizable potential. Detection limits obtained from 0.10 pmol injection, at a S/N of 3, were 4.0–12.7 fmol. The mean intra‐ and inter‐assay precision for all aliphatic amine levels were <3.84 and 3.21%, respectively. Excellent linear responses were observed with coefficients of >0.9995.     

Determination of thiol compounds by HPLC and fluorescence detection with 1,3,5,7‐tetramethyl‐8‐bromomethyl‐difluoroboradiaza‐s‐indacene

Altered levels of thiols in biological fluids are considered to be an important indicator for several diseases. In this article, 1,3,5,7‐tetramethyl‐8‐bromomethyl‐difluoroboradiaza‐s‐indacene is proposed as a fluorescent derivatization reagent for the determination of thiols including glutathione, cysteine, N‐acetylcysteine, and homocysteine by HPLC. Under the optimized derivatization and separation conditions, a baseline separation of all the four derivatives has been achieved using isocratic elution on an RP C₈ column within 26 min. With fluorescence detection at 505 and 525 nm for the excitation and emission, respectively, the LODs (S/N = 3) are from 0.2 nM (glutathione) to 0.8 nM (cysteine). The feasibility of this method in real samples has been evaluated by the determination of thiols in human plasma from the healthy persons and hypertensive patients with recoveries of 92–105.3%.     

Simple determination of betaine,l‐carnitine and choline in human urine using self‐packed column and column‐switching ion chromatography with nonsuppressed conductivity detection

A sequential online extraction, clean‐up and separation system for the determination of betaine, l ‐carnitine and choline in human urine using column‐switching ion chromatography with nonsuppressed conductivity detection was developed in this work. A self‐packed pretreatment column (50 × 4.6 mm, i.d.) was used for the extraction and clean‐up of betaine, l ‐carnitine and choline. The separation was achieved using self‐packed cationic exchange column (150 × 4.6 mm, i.d.), followed by nonsuppressed conductivity detection. Under optimized experimental conditions, the developed method presented good analytical performance, with excellent linearity in the range of 0.60–100 μg mL⁻¹ for betaine, 0.75–100 μg mL⁻¹ for l ‐carnitine and 0.50–100 μg mL⁻¹ for choline, with all correlation coefficients (R²) >0.99 in urine. The limits of detection were 0.15 μg mL⁻¹ for betaine, 0.20 μg mL⁻¹ for l ‐carnitine and 0.09 μg mL⁻¹ for choline. The intra‐ and inter‐day accuracy and precision for all quality controls were within ±10.32 and ±9.05%, respectively. Satisfactory recovery was observed between 92.8 and 102.0%. The validated method was successfully applied to the detection of urinary samples from 10 healthy people. The values detected in human urine using the proposed method showed good agreement with the measurement reported previously.     

A validated stability‐indicating ultra performance liquid chromatography method for the determination of potential process‐related impurities in eplerenone

A simple, sensitive, and accurate stability‐indicating analytical method has been developed and validated using ultra high performance liquid chromatography. The developed method is used to evaluate the related substances of eplerenone (EP). The degradation behavior of EP under stress conditions was determined, and the major degradants were identified by ultra high performance liquid chromatography with tandem mass spectrometry. The chromatographic conditions were optimized using an impurity‐spiked solution, and the samples, generated from forced degradation studies. The resolution of EP, its potential impurities, and its degradation products was performed on a Waters UPLC BEH C₁₈ column (50 × 2.1 mm, 1.7 μm) by linear gradient elution using a mobile phase consisting of 10 mmol/L ammonium acetate adjusted to pH 4.5, methanol and acetonitrile. A photo‐diode array detector set at 245 nm was used for detection. The flow rate was set at 0.3 mL/min. The procedure had good specificity, linearity (0.02–3.14 μg/mL), recovery (96.1–103.9%), limit of detection (0.01–0.02 μg/mL), limit of quantitation (0.03–0.05 μg/mL), and robustness. The correction factors of the process‐related substances were calculated.     

High‐efficiency sample preparation approach to determine acrylamide levels in high‐fat foods

An improved sample preparation method was developed to enhance acrylamide recovery in high‐fat foods. Prior to concentration, distilled deionized water was added to protect acrylamide from degradation, resulting in a higher acrylamide recovery rate from fried potato chips. A Chrome‐Matrix C₁₈ column (2.6 μm, 2.1 × 100 mm) was used for the first time to analyze acrylamide levels using ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry, displaying good separation of acrylamide from interference. A solid‐phase extraction procedure was avoided, and an average recovery of >89.00% was achieved from different food matrices for three different acrylamide spiking levels. Good reproducibility was observed, with an intraday relative standard deviation of 0.04–2.38%, and an interday relative standard deviation of 2.34–3.26%. Thus, combining the improved sample preparation method for acrylamide analysis with the separation on a Chrome‐Matrix C₁₈ column (2.6 μm, 2.1 × 100 mm) using ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry is highly useful for analyzing acrylamide levels in complex food matrices.     

Determination of betaine,l‐carnitine,and choline in human urine using a self‐packed column and column‐switching ion chromatography with nonsuppressed conductivity detection

A simple method for the determination of betaine, l ‐carnitine, and choline in human urine was developed based on column‐switching ion chromatography coupled with nonsuppressed conductivity detection by using a self‐packed column. A pretreatment column (50 mm × 4.6 mm, id) packed with poly(glycidyl methacrylate‐divinylbenzene) microspheres was used for the extraction and cleanup of analytes. Chromatographic separation was achieved within 10 min on a cationic exchange column (150 mm × 4.6 mm, id) using maleic anhydride modified poly(glycidyl methacrylate‐divinylbenzene) as the particles for packing. The detection was performed by ion chromatography with nonsuppressed conductivity detection. Parameters including column‐switching time, eluent type, flow rates of eluent, and interfering effects were optimized. Linearity (r ² ≥ 0.99) was obtained for the concentration range of 0.50–100, 0.75–100, and 0.25–100 μg/mL for betaine, l ‐carnitine, and choline, respectively. Detection limits were 0.12, 0.20, and 0.05 μg/mL for betaine, l ‐carnitine, and choline, respectively. The intra‐ and interday accuracy and precision for all quality controls were within ±10.11%. Satisfactory recovery was observed between 92.5 and 105.0%. The validated method was successfully applied for the determination of betaine, l ‐carnitine, and choline in urine samples from healthy people.