Quantitative determination of risperidone,paliperidone and olanzapine in human serum by liquid chromatography–tandem mass spectrometry coupled with on‐line solid‐phase extraction

A recent guideline recommends therapeutic drug monitoring for risperidone, paliperidone and olanzapine, which are frequently used second‐generation antipsychotics. We developed a simple high‐performance liquid chromatography–tandem mass spectrometry coupled with an online solid‐phase extraction method that can be used to measure risperidone, paliperidone and olanzapine using small (40 μL) samples. The analytes were extracted from serum samples automatically pre‐concentrated and purified by C₈ (5 μm, 2.1 × 30 mm) solid‐phase extraction cartridges, then chromatographed on an Xbidge™ C₁₈ column (3.5 μm, 100 × 2.1 mm) thermostatted at 30°C with a mobile phase consisting of 70% acetonitrile and 30% ammonium hydroxide 1% solution at an isocratic flow rate of 0.3 mL/min, and detected with tandem mass spectrometry. The assay was validated in the concentration range from 2.5 to 160 ng/mL. Intra‐ and inter‐day precision for all analytes was between 1.1 and 8.2%; method accuracy was between 6.6 and 7.6%. The risperidone and paliperidone assay was compared with a high‐performance liquid chromatography‐ultraviolet assay currently used in our hospital for risperidone and paliperidone therapeutic drug monitoring, and the results of weighted Deming regression analysis showed good agreement. For the olanzapine assay, we compared 20 samples in separate re‐assays on different days; all the relative errors were within the 20% recommended limit.     

Determination of trimethylamine‐N‐oxide in combination with l‐carnitine and γ‐butyrobetaine in human plasma by UPLC/MS/MS

An ultra‐high‐performance liquid chromatography–mass spectrometry (UPLC/MS/MS) method was developed and validated for the quantification of trimethylamine‐N‐oxide (TMAO) simultaneously with TMAO‐related molecules l ‐carnitine and γ‐butyrobetaine (GBB) in human blood plasma. The separation of analytes was achieved using a Hydrophilic interaction liquid chromatography (HILIC)‐type column with ammonium acetate–acetonitrile as the mobile phase. TMAO determination was validated according to valid US Food and Drug Administration guidelines. The developed method was successfully applied to plasma samples from healthy volunteers. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and comparison of liquid–liquid extraction and accelerated solvent extraction methods for quantitative analysis of chloramphenicol,thiamphenicol, florfenicol,and florfenicol amine in poultry eggs

Accelerated solvent extraction was investigated as a novel alternative technology for the separation and quantitative analysis of chloramphenicol, thiamphenicol, florfenicol, and florfenicol amine from poultry eggs, and the results were compared with the results of liquid–liquid extraction. Rapid quantification of the target compounds was carried out by ultra‐performance liquid chromatography‐electrospray ionization tandem triple quadrupole mass spectrometry. This optimized method was validated according to the requirements defined by the European Union and the United States Food and Drug Administration. Finally, the new approach was successfully applied to the quantitative determination of these analytes in 90 commercial poultry eggs from local supermarkets.     

Simultaneous determination of four secoiridoid and iridoid glycosides in rat plasma by ultra performance liquid chromatography–tandem mass spectrometry and its application to a comparative pharmacokinetic study

A simple, reliable and rapid ultra‐performance liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous quantification of four secoiridoid (gentiopicroside, swertiamarin, sweroside) and iridoid glycosides (loganic acid), the bio‐active ingredients in rat plasma. After liquid–liquid extraction, chromatographic separation was accomplished on a Shim‐pack XR‐ODS column with a mobile phase consisting of methanol and 0.1% formic acid in water. A triple quadrupole tandem mass spectrometry equipped with an electrospray ionization source was used as detector operating both in positive and negative ionization mode and operated by multiple‐reaction monitoring scanning. The lower limits of quantitation were 0.25–30 ng/mL for all the analytes. Both intra‐day and inter‐day precision and accuracy of analytes were well within acceptance criteria (±15%). The mean extraction recoveries of analytes and internal standard (amygdalin) from rat plasma were all >71.4%. The validated method was successfully applied to a comparative pharmacokinetic study of four analytes in rat plasma between normal and arthritic rats after oral administration of Huo Luo Xiao Ling Dan and Gentiana macrophylla extract, respectively. Results showed significant differences in pharmacokinetic properties of the analytes among the different groups. Copyright © 2015 John Wiley & Sons, Ltd.     

Automatic on‐line solid‐phase extraction with ultra‐high performance liquid chromatography and tandem mass spectrometry for the determination of ten antipsychotics in human plasma

An automatic on‐line solid‐phase extraction with ultra‐high performance liquid chromatography and tandem mass spectrometry method was developed for the simultaneous determination of ten antipsychotics in human plasma. The plasma sample after filtration was injected directly into the system without any pretreatment. A Shim‐pack MAYI‐C₈ (G) column was used as a solid‐phase extraction column, and all the analytes were separated on a Shim‐pack XR‐ODS III column with a mobile phase consisting of 0.1% v/v formic acid in water with 5 mM ammonium acetate and acetonitrile. The method features were systematically investigated, including extraction conditions, desorption conditions, the equilibration solution, the valve switching time, and the dilution for column‐head stacking. Under the optimized conditions, the whole analysis procedure took only 10 min. The limits of quantitation were in the range of 0.00321–2.75 μg/L and the recoveries ranged from 75.9 to 122%. Compared with the off‐line ultra‐high performance liquid chromatography and the reported methods, this validated on‐line method showed significant advantages such as minimal pretreatment, shortest analysis time, and highest sensitivity. The results indicated that this automatic on‐line method was rapid, sensitive, and reliable for the determination of antipsychotics in plasma and could be extended to other target analytes in biological samples.     

Pharmacokinetic comparison of five tanshinones in normal and arthritic rats after oral administration of Huo Luo Xiao Ling Dan or its single herb extract by UPLC‐MS/MS

A fast, sensitive and reliable ultra performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) method has been developed and validated for simultaneous quantitation and pharmacokinetic study of five tanshinones (tanshinone I, tanshinone IIA, tanshinone IIB, dihydrotanshinone I, cryptotanshinone), the bio‐active ingredients of Huo Luo Xiao Ling Dan (HLXLD) in rat plasma. After liquid–liquid extraction, chromatographic separation was accomplished on a Shim‐pack XR‐ODS column (75 × 3.0 mm, 2.2 µm particles) and eluted with a mobile phase consisting of acetonitrile–0.05% formic acid aqueous solution (80:20, v/v) at a flow rate of 0.4 mL/min, and the total run time was 7.0 min. The detection was performed on a triple quadrupole tandem mass spectrometry equipped with an electrospray ionization source in positive ionization and multiple reaction monitoring mode. The lower limits of quantification were 0.050–0.400 ng/mL for all the analytes. Linearity, precision and accuracy, the mean extraction recoveries and matrix effects all satisfied criteria for acceptance. This validated method was successfully applied to a comparative pharmacokinetic study of five bio‐active components in rat plasma after oral administration of HLXLD or Salvia miltiorrhiza extract in normal and arthritic rats. The results showed that there were different pharmacokinetic characteristics among different groups. Copyright © 2016 John Wiley & Sons, Ltd.     

Simultaneous quantification of fentanyl,sufentanil, cefazolin,doxapram and keto‐doxapram in plasma using liquid chromatography–tandem mass spectrometry

A simple and specific UPLC–MS/MS method was developed and validated for simultaneous quantification of fentanyl, sufentanil, cefazolin, doxapram and its active metabolite keto‐doxapram. The internal standard was fentanyl‐d5 for all analytes. Chromatographic separation was achieved with a reversed‐phase Acquity UPLC HSS T3 column with a run‐time of only 5.0 min per injected sample. Gradient elution was performed with a mobile phase consisting of ammonium acetate or formic acid in Milli‐Q ultrapure water or in methanol with a total flow rate of 0.4 mL min⁻¹. A plasma volume of only 50 μL was required to achieve adequate accuracy and precision. Calibration curves of all five analytes were linear. All analytes were stable for at least 48 h in the autosampler. The method was validated according to US Food and Drug Administration guidelines. This method allows quantification of fentanyl, sufentanil, cefazolin, doxapram and keto‐doxapram, which is useful for research as well as therapeutic drug monitoring, if applicable. The strength of this method is the combination of a small sample volume, a short run‐time, a deuterated internal standard, an easy sample preparation method and the ability to simultaneously quantify all analytes in one run.     

Fast and sensitive analysis of beta blockers by ultra‐high‐performance liquid chromatography coupled with ultra‐high‐resolution TOF mass spectrometry

This paper presents a method for the determination of acebutolol, betaxolol, bisoprolol, metoprolol, nebivolol and sotalol in human serum by liquid–liquid extraction and ultra‐high‐performance liquid chromatography coupled with ultra‐high‐resolution TOF mass spectrometry. After liquid–liquid extraction, beta blockers were separated on a reverse‐phase analytical column (Acclaim RS 120; 100 × 2.1 mm, 2.2 μm). The total run time was 6 min for each sample. Linearity, limit of detection, limit of quantification, matrix effects, specificity, precision, accuracy, recovery and sample stability were evaluated. The method was successfully applied to the therapeutic drug monitoring of 108 patients with hypertension. This method was also used for determination of beta blockers in 33 intoxicated patients.     

Quantitation of acotiamide in rat plasma by UHPLC‐Q‐TOF‐MS: method development,validation and application to pharmacokinetics

A novel, sensitive and selective ultra‐high‐performance liquid chromatography–electrospray ionization mass spectrometry method was developed and validated for the quantification of acotiamide (ACT), a first‐in‐class drug used in functional dyspepsia, in rat plasma. A simple protein precipitation method with acetonitrile as precipitating solvent was used to extract ACT from rat plasma. ACT and an internal standard (mirabegron, IS) were separated on an Agilent poroshell EC C₁₈ column (50 × 3.0 mm, 2.7 µm) using methanol–10 mM ammonium acetate binary gradient mobile phase at a flow rate of 0.4 mL/min over 4 min run time. Detection was performed using target ions of [M + H]⁺ at m/z 451.2010 for ACT and m/z 397.1693 for IS in selective ion mode. The method was validated in the calibration range of 1.31–1000 ng/mL. All the validation parameters were well within the limits. The method demonstrated good performances in terms of intra‐ and inter‐day precision (3.27–12.60% CV) and accuracy (87.96–104.94%). Thus the present ultra‐high‐pressure liquid chromatograhy–high‐resolution mass spectrometry method for determination of ACT in rat plasma, is highly sensitive and rapid with a short run‐time of 4 min, can be suitable for high sample throughput and for large batches of biological samples in pharmacokinetic studies. This method can be extended to measure plasma concentrations of ACT in humans to understand drug metabolism, drug interaction and adverse effects. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and validation of an ultra‐high‐performance liquid chromatography–tandem mass spectrometry method to determine the bioavailability of warfarin and its major metabolite 7‐hydroxy warfarin in rats dosed with oral formulations containing different polymorphic forms

A simple, sensitive and rapid ultra‐high‐performance liquid chromatography–electrospray ionization–tandem mass spectrometry method was developed and validated for the quantification of warfarin and 7‐hydroxy warfarin in Sprague Dawley (SD) rats. Animals were administered a single dose of warfarin sodium formulations (crystalline and amorphous) at 12 mg/kg via oral gavage and blood was drawn over a 96‐h time course. Sample process recoveries, matrix effect and analyte stability were determined. The linearity for warfarin and 7‐hydroxy warfarin was from 5 to 2000 ng/mL in blank SD rat plasma. Correlation coefficients (r²) for standard calibration curves were >.98 and analytes quantified within ±15% of target at all calibrator concentrations. The average percent accuracy and precision for intra‐ and inter‐day were 93.7%–113.8% and ≤12.1%, respectively, for warfarin and 7‐hydroxy warfarin, across the quality control standards (5, 10, 500, 1800 and 2000 ng/mL). Acceptable analytical recovery (>55%) was achieved with process efficiencies >41.5% and matrix effects <139.9% over the analytical range. Both analytes were stable in stock solution, autosampler, benchtop and three cycles of freeze–thaw with percent accuracy ≥90.2% and precision (percent relative standard deviation) ≤14%. The validated method was successfully applied to a pre‐clinical bioavailability study of crystalline and amorphous warfarin sodium formulations in SD rats.     

Simultaneous determination of aurantio‐obtusin,chrysoobtusin, obtusin and 1‐desmethylobtusin in rat plasma by UHPLC‐MS/MS

A sensitive and reliable ultra‐high‐performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UHPLC‐MS/MS) method was developed and validated for the simultaneous determination of four active components of Semen Cassiae extract (aurantio‐obtusin, chrysoobtusin, obtusin and 1‐desmethylobtusin) in rat plasma after oral administration. Chromatographic separation was achieved on an Agilent Poroshell 120 C₁₈ column with gradient elution using a mobile phase that consisted of acetonitrile‐ammonium acetate in water (30 mm ) at a flow rate of 0.4 mL/min. Detection was performed by a triple‐quadrupole tandem mass spectrometer in multiple reaction monitoring mode. The calibration curve was linear over a range of 3.24–1296 ng/mL for aurantio‐obtusin, 0.77–618 ng/mL for chrysoobtusin, 34.55–1818 ng/mL for obtusin and 1.86–1485 ng/mL for 1‐desmethylobtusin. Inter‐ and intra‐day assay variation was <15%. All analytes were shown to be stable during all sample storage and analysis procedures. Copyright © 2013 John Wiley & Sons, Ltd.     

Comparative pharmacokinetic study of the components of Jia‐Wei‐Kai‐Xin‐San in normal and vascular dementia rats by ultra‐fast liquid chromatography coupled with tandem mass spectrometry

A fast, sensitive, and reliable ultra‐high performance liquid chromatography coupled with tandem mass spectrometry method has been developed and validated for simultaneous quantification of geniposide, polygalaxanthone III, 3,6′‐disinapoyl sucrose, α‐asarone, β‐asarone, poricoic acid A, poricoic acid B, dehydrotumulosic acid, deoxyschizandrin, schizandrin B, and kaempferide in plasma after oral administration of extracts of Jia‐Wei‐Kai‐Xin‐San in normal and vascular dementia rats. The developed method was precise and accurate within the linearity range of the analytes. The lower limits of quantification were 1.04–2.68 ng/mL for all the analytes. Both intra‐ and inter day precision and accuracy of the analytes were all within accepted criteria. The mean extraction recoveries of the analytes and the internal standard from rat plasma were all >60.0%. The validated method had been successfully applied to compare pharmacokinetic profiles of the analytes in plasma of normal and vascular dementia rat treated with herbal extracts. Results indicated that differences existed between normal and vascular dementia model rats except dehydrotumulosic acid and kaempferide, which might be due to the pathology of vascular dementia and pharmacological effect of the analytes. These pharmacokinetic studies might benefit for the mechanism exploration and clinical use of traditional Chinese medicine formulae.     

Simultaneous analysis of 11 main active components in Cirsium setosum based on HPLC‐ESI‐MS/MS and combined with statistical methods

A novel method based on high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry was developed for simultaneous determination of the 11 major active components including ten flavonoids and one phenolic acid in Cirsium setosum. Separation was performed on a reversed‐phase C₁₈ column with gradient elution of methanol and 0.1‰ acetic acid (v/v). The identification and quantification of the analytes were achieved on a hybrid quadrupole linear ion trap mass spectrometer. Multiple‐reaction monitoring scanning was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run. Full validation of the assay was carried out including linearity, precision, accuracy, stability, limits of detection and quantification. The results demonstrated that the method developed was reliable, rapid, and specific. The 25 batches of C. setosum samples from different sources were first determined using the developed method and the total contents of 11 analytes ranged from 1717.460 to 23028.258 μg/g. Among them, the content of linarin was highest, and its mean value was 7340.967 μg/g. Principal component analysis and hierarchical clustering analysis were performed to differentiate and classify the samples, which is helpful for comprehensive evaluation of the quality of C. setosum.     

Studies on excretion kinetics of ten constituents in rat urine after oral administration of Shensong Yangxin Capsule by UPLC‐MS/MS

A rapid and sensitive ultra‐high performance liquid chromatography–mass spectrometry (UPLC‐MS/MS) method was developed and validated for the quantification of 10 major active constituents in rat urine after oral administration of Shensong Yangxin Capsule (SSYX) using diazepam as an internal standard (IS). The urine samples were pretreated and extracted by solid‐phase extraction prior to UPLC. Chromatographic separation was achieved on a Waters C₁₈ (2.1 × 50 mm, 1.7 µm) column using a gradient elution program with 0.1% formic acid aqueous solution and acetonitrile at a flow rate of 0.4 mL/min. Detection and quantitation were accomplished by a hybrid quadrupole mass spectrometer using electrospray ionization source and multiple reaction monitoring in the positive ionization mode. The mass transition ion‐pairs (m/z) for quantitation were all optimized and the total run time was 4.50 min. The specificity, linearity, accuracy, precision, recovery, matrix effect and stabilities were all validated for the analytes in urine samples. The validation results indicated that this method was simple, rapid, specific and reliable. The proposed method was successfully applied to investigate the urinary excretion kinetics of 10 compounds in rat after oral administration of SSYX. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantification of midazolam,morphine and metabolites in plasma using 96‐well solid‐phase extraction and ultra‐performance liquid chromatography–tandem mass spectrometry

Currently, pharmacokinetic–pharmacodynamic studies of sedatives and analgesics are performed in neonates and children to find suitable dose regimens. As a result, sensitive assays using only small volumes of blood are necessary to determine drug and metabolite concentrations. We developed an ultra‐performance liquid chromatographic method with tandem mass spectrometry detection for quantification of midazolam, 1‐hydroxymidazolam, hydroxymidazolamglucuronide, morphine, morphine‐3‐glucuronide and morphine‐6‐glucuronide in 100 μL of plasma. Cleanup consisted of 96 wells micro‐solid phase extraction, before reversed‐phase chromatographic separation (ultra‐performance liquid chromatography) and selective detection using electrospray ionization tandem mass spectrometry. Separate solid‐phase extraction methods were necessary to quantify morphine, midazolam and their metabolites because of each group's physicochemical properties. Standard curves were linear over a large dynamic range with adequate limits of quantitation. Intra‐ and interrun accuracy and precision were within 85–115% (of nominal concentration using a fresh calibration curve) and 15% (coefficient of variation, CV) respectively. Recoveries were >80% for all analytes, with interbatch CVs (as a measure of matrix effects) of less than 15% over six batches of plasma. Stability in plasma and extracts was sufficient, allowing large autosampler loads. Runtime was 3.00 min per sample for each method. The combination of 96‐well micro‐SPE and UPLC‐MS/MS allows reliable quantification of morphine, midazolam and their major metabolites in 100 μL of plasma. Copyright © 2010 John Wiley & Sons, Ltd.     

Ultra‐high pressure liquid chromatography–tandem mass spectrometry method for the determination of omarigliptin in rat plasma and its application to a pharmacokinetic study in rats

Omarigliptin is a novel long‐acting dipeptidyl peptidase‐4 inhibitor used for the treatment of type 2 diabetes. In this work, a sensitive and selective ultra‐high pressure liquid chromatography tandem mass spectrometry method was developed and validated for the determination of omarigliptin in rat plasma. Sample preparation was performed by protein precipitation with acetonitrile. Chromatographic separation of analytes was achieved on an RRHD Eclipse Plus C18 column (2.1 × 50 mm, 1.8 μm), using gradient mobile phase (0.1% formic acid–acetonitrile) at a flow rate of 0.4 mL/min. Detection was performed in multiple reaction monitoring mode, with target fragment ions m/z 399.1 → 152.9 for omarigliptin and m/z 237.1 → 194 for the internal standard. The total run time was 4 min. Retention time of omarigliptin and internal standard was 1.25 and 2.12 min, respectively. Relative standard deviation (%) of the intra‐ and inter‐day precision was below 10.0%, and accuracy was between 97.9% and 105.3%. Calibration curve was established over the range 2–5000 ng/mL with good linearity. The lower limit of quantification and limit of detection of omarigliptin were 2 and 0.25 ng/mL, respectively. Mean recoveries were in the range 87.3–95.1% for omarigliptin. No matrix effect was observed in this method. This novel method has been successfully applied to a pharmacokinetic study of omarigliptin in rats. The absolute bioavailability of omarigliptin was identified as high as 87.31%.     

UPLC–MS/MS determination of flavokawain B,a novel anti‐tumor chemotherapeutic agent in rat plasma and its application to a pharmacokinetic study in rats

A sensitive, selective and rapid ultra‐performance liquid chromatography/tandem mass spectrometry method was developed and validated for the quantification of flavokawain B in rat plasma using myrislignan as an internal standard. Sample preparation was accomplished through a protein precipitation extraction process. Chromatographic resolution of flavokawain B and the IS was achieved on an Agilent XDB‐C₁₈ column (2.1 × 100 mm, 1.8 μm) using a gradient mobile phase comprising 0.1% formic acid in water and acetonitrile delivered at a flow rate of 0.5 mL/min. Flavokawain B and the IS eluted at 3.27 and 1.96 min, respectively. The total chromatographic run time was 6.0 min. A linear response function was constructed in the concentration range 0.524–1048 ng/mL. Method validation was performed as per the US Food and Drug Administration guidelines and the results met the acceptance criteria. Intra‐ and inter‐day accuracy and precision were in the ranges of ?14.3–13.2 and 3.4–11.8%, respectively. Flavokawain B was demonstrated to be stable under various stability conditions. This method has been applied to a pharmacokinetic study in rats.     

Simultaneous quantification and semi‐quantification of amentoflavone and its metabolites in human intestinal bacteria by liquid chromatography Orbitrap high‐resolution mass spectrometry

A quick, easy, effective method followed by ultra‐high‐pressure liquid chromatography coupled with linear ion trap–Orbitrap tandem mass spectrometry (UHPLC‐LTQ‐Orbitrap MS) was developed for the simultaneous identification and quantification of the metabolites produced by amentoflavone (AMF) in human intestinal bacteria from human feces. The method validated for quantification of AMF concerning precision, accuracy, recovery, matrix effect, stability and limits showed acceptable results. Compared with blank human intestinal bacteria chromatography, three metabolites were identified based on high‐accuracy protonated precursors and multi‐stage mass spectrometry (MSⁿ ) using the proposed strategy. At the same time, a new method was developed for semi‐quantification of three metabolites. We describe the trend over 24 h of concentration–time curves for AMF and its metabolites. Moreover, the main metabolic pathway of AMF was clarified in human intestinal bacteria. The method was validated and successfully applied to the detection and quantification of AMF and its metabolites.     

A fast and simple approach for the quantification of 40 illicit drugs,medicines, and pesticides in blood and urine samples by UHPLC‐MS/MS

A fast and simple approach to overcome challenges in emergency toxicological analysis, using ultra‐high performance liquid chromatography–tandem mass spectrometry (UHPLC‐MS/MS) has been developed, for the detection of analytes in blood and urine samples from the following drug classes: analgesics, benzodiazepines, antidepressants, anticonvulsants, drugs of abuse, and pesticides. These substances are relevant in the context of emergency toxicology in Brazil. The sample preparation procedure was relatively easy and fast to perform. The method was fully validated giving limits of in the range of 0.5 and 20 ng mL^?1 for blood and urine samples. The intraday and interday precision and accuracy were considered adequate for all analytes once the relative standard deviation (RSD) (%) was lower than 20% for quality control (QC) low and lower than 15% for CQ medium and high. The developed method was successfully applied to 320 real samples collected at the Poison Control Center of São Paulo, and 89.1% have shown to be positive for some of the analytes. This confirms its applicability and importance to emergency toxicological analysis, and it could be very useful in both fields of clinical and forensic toxicology.     

Simultaneous determination of l‐tetrahydropalmatine and its active metabolites in rat plasma by a sensitive ultra‐high‐performance liquid chromatography with tandem mass spectrometry method and its application in a pharmacokinetic study

A sensitive and reliable ultra‐high‐performance liquid chromatography with tandem mass spectrometry (UHPLC–MS/MS) method was developed and validated for simultaneous determination of l ‐tetrahydropalmatine (l ‐THP) and its active metabolites l ‐isocorypalmine (l ‐ICP) and L ‐corydalmine (l ‐CD) in rat plasma. The analytes were extracted by liquid–liquid extraction and separated on a Bonshell ASB C₁₈ column (2.1 × 100 mm; 2.7 μm; Agela) using acetonitrile–formic acid aqueous as mobile phase at a flow rate of 0.2 mL/min in gradient mode. The method was validated over the concentration range of 4.00–2500 ng/mL for l ‐THP, 0.400–250 ng/mL for l ‐ICP and 1.00–625 ng/mL for l ‐CD. Intra‐ and inter‐day accuracy and precision were within the acceptable limits of <15% at all concentrations. Correlation coefficients (r ) for the calibration curves were >0.99 for all analytes. The quantitative method was successfully applied for simultaneous determination of l ‐THP and its active metabolites in a pharmacokinetic study after oral administration with l ‐THP at a dose of 15 mg/kg to rats.