High-performance liquid chromatographic–tandem mass spectrometric determination of 3-hydroxypropylmercapturic acid in human urine

A sensitive and specific high-performance liquid chromatographic–tandem mass spectrometric (HPLC–MS–MS) method was developed for the determination of 3-hydroxypropylmercapturic acid (3-HPMA) in human urine. Samples were extracted using ENV+ cartridges and then injected onto a C8 Superspher Select B column with acetonitrile and formic acid as eluent (5:95, v/v). N-Acetylcysteine was used as internal standard for HPLC–MS–MS. Linearity was given in the tested range of 50–5000 ng/ml urine. The limit of quantification was 50 ng/ml. Precision, as C.V., in the tested range of 50–5000 ng/ml was 1.47–6.04%. Accuracy ranged from 87 to 114%. 3-HPMA was stable in human urine at 37°C for 24 h. The method was able to quantify 3-HPMA in urine of non-smokers and smokers.     

Simple liquid chromatography method for the rapid simultaneous determination of prednisolone and cortisol in plasma and urine using hydrophilic lipophilic balanced solid phase extraction cartridges

This article describes the development and validation of a simple solid phase extraction (SPE) and HPLC method for the extraction and the specific determination of prednisolone and hydrocortisone (cortisol) in both plasma and urine using one washing step with Oasis hydrophilic lipophilic balanced (HLB) cartridges (1 ml/30 mg, 30 microm). Recoveries of prednisolone and cortisol from plasma and urine exceeded 82%. The limit of quantification (LOQ) in plasma and urine was 9.9 and 6.7 ng/ml for cortisol, respectively, and 11.6 and 8.0 ng/ml for prednisolone, respectively. The intraday and interday precision (measured by CV%) for both prednisolone and cortisol in both plasma and urine was always less than 7%. The accuracy (measured by relative error %) for both prednisolone and cortisol in both plasma and urine was always less than 8%. The advantages of the developed method are the use of a one step washing SPE utilising HLB cartridges which do not suffer the drying out problems of conventional SPE cartridges and the time saving when compared with solvent extraction (SE), in addition to the simultaneous determination of prednisolone and cortisol in both plasma and urine.     

A new method for the quantitation of propofol in human plasma: efficient solid-phase extraction and liquid chromatography/APCI-triple quadrupole mass spectrometry detection

Propofol (2,6-diisopropyl phenol) is widely used for the induction and maintenance of anesthesia. Analyses of its pharmacokinetics require simple and sensitive methods for quantitation of propofol in human plasma. Previously reported HPLC and GC methods are limited by cumbersome extraction steps. We describe a novel method that combines sample preparation by solid-phase extraction (SPE) with hydrophilic-lipophilic balance cartridges and analysis with a sensitive LC-APCI-triple quadrupole mass spectrometry (MS/MS) method for better quantitation. The absolute recovery of the analyte was greater than 96%. The limit of quantification for propofol in plasma at a signal-to-noise ratio of 10 was 5 ng/ml. The precision of the assay yielded coefficients of variation ranging from 2.9 to 5.3% and an accuracies of 99-105%. Our method advances the quantitative analysis of propofol in human plasma by combining simple, rapid and efficient SPE with specific and sensitive quantitation by HPLC with APCI-MS/MS detection.     

Measurement of plasma 5-hydroxyindole acetic acid by liquid chromatography tandem mass spectrometry—Comparison with HPLC methodology

In patients with carcinoid disease, urinary concentration of the serotonin metabolite 5-hydroxyindole acetic acid (5-HIAA) is currently used to monitor disease progression or response to treatment as it is the metabolic end-product resulting from free and stored serotonin turnover. However, due to the undignified, cumbersome and error-prone nature of 24-h urine collections, there is constant pressure to replace them. It has been demonstrated using high performance liquid chromatography (HPLC) with fluorescence detection technology that plasma can achieve this, with the added advantage that it can be used for diagnostic purposes also. Here we describe a much simpler method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) that is twice as fast as a HPLC method currently in routine use. The sample preparation protocol requires 50 μL of plasma and a simple protein precipitation step facilitated by acetonitrile. Chromatography was performed on a Phenomenex C18 Security Guard? column coupled to a SIELC Primesep B reversed-phase, anion-exchange dual chemistry column and methanolic mobile phase gradient elution. Eluant was directly connected to a Waters^® Quattro Premier? XE tandem mass spectrometer operating in positive ion mode. We detected multiple reaction monitoring transitions m/z 191.9 > 145.6 and 193.9 > 147.6 for 5-HIAA and d2-5-HIAA respectively, which co-eluted at 2.1 min. Ion suppression was negligible, recovery from spiked plasma was 103% (range 97–113%) and the method showed good linearity to 10,000 nmol/L (r² = 0.999). Within-batch and between-batch imprecision was <10% and bias <15% at 3 concentrations, the limit of detection was 5 nmol/L and lower limit of quantitation 15 nmol/L. No interference was observed with l-tryptophan or 5-hydroxytryptamine. Comparison of LC–MS/MS and HPLC showed good agreement between the two methods but this LC–MS/MS assay displays several advantages; it requires 10-fold less sample, has a simpler extraction procedure and extended linearity, thus increasing laboratory throughput, lowering reagent costs and removing the need to dilute samples in patients with established carcinoid disease being monitored for therapeutic efficacy.     

Measurement of succinylcholine concentration in human plasma by electrospray tandem mass spectrometry

An electrospray mass spectrometric method for the quantification of the depolarizing neuromuscular blocking agent succinylcholine (SUX) is described. An extraction method compatible with direct infusion inlet was developed and leads to an analysis cycle time of 7--8 min instead of 25 min that would be required for HPLC inlet. SUX was extracted from human plasma on C1 solid-phase cartridges and was analyzed using positive ion electrospray tandem mass spectrometry (ESI-MS/MS). SUX plasma concentrations were determined by a stable isotope dilution assay using hexadeuterosuccinylcholine diiodide (SUXd6) as the internal standard. The calibration curve was prepared using the ratio of intensities of the major product ions in the collision-induced dissociation spectrum for known concentration ratios of SUX and SUXd6 in plasma. Calibration curves for the quantification were linear from 25 to 4000 ng/ml. For intraday precision, CV were < or =6% and accuracy ranged from 98 to 103%. For the interday precision, CV were < or =10% and accuracy ranged from 90 to 102%. This method is specific, sensitive, reproducible, and practical in a clinical setting.     

Quantitative analysis of heterocyclic amines in urine by liquid chromatography coupled with tandem mass spectrometry

A sensitive, reproducible, and rapid analytical method for the analysis of trace-level heterocyclic amines (HCAs) that are expected to have high levels of human exposure was developed. Liquid–liquid extraction (LLE) with dichloromethane (DCM) followed by solid-phase extraction (SPE) was carried out. Liquid extraction with DCM under basic conditions was efficient in extracting HCAs from urine samples. For further purification, mixed mode cationic exchange (MCX) cartridges were applied to eliminate the remaining interferences after liquid extraction. Separation and quantification were performed by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) in selected reaction monitoring (SRM) mode. The overall recoveries ranged between 71.0% and 113.6% with relative standard deviations (RSDs) of 5.1% to 14.7% for the entire procedure. The limits of detection (LODs) and limits of quantification (LOQs) of the proposed analytical method were in the ranges of 0.04 to 0.10 ng/ml and 0.15 to 0.36 ng/ml, respectively. This method was applied to the analysis of monitoring in urine samples for Korean school children, and the results demonstrated that the method can be used for the trace determination of HCAs in urine samples.     

LC/MS/MS measurement of penicillin G in bovine plasma, urine, and biopsy samples taken from kidneys of standing animals

Methods for the measurement of penicillin concentration in bovine plasma, kidney and urine were developed and validated. Detection was based on liquid chromatography/tandem mass spectrometry (LC/MS/MS). Phenethecillin was used as an internal standard. Plasma was extracted with acetonitrile using a method with a calculated limit of quantitation (LOQ) of 12 ng/mL. Kidney samples were homogenized in water and acetonitrile, then cleaned up on C18-bonded silica SPE cartridges. The LOQ of this procedure was 10 ng/g. Urine samples were diluted, filtered, and analyzed directly. The LOQ of this procedure was 63 ng/mL. The overall accuracy for plasma was 103% with coefficient of variation (CV) of 3%; for kidney, 96% and 11%, respectively, and for urine, 98% and 4%, respectively. These methods were applied to the analysis of plasma, urine, and kidney biopsy samples taken from standing animals that had been dosed with penicillin.     

Assessment of potential (inhalation and dermal) and actual exposure to acetamiprid by greenhouse applicators using liquid chromatography-tandem mass spectrometry

New analytical methods based on liquid chromatography with electrospray tandem mass spectrometry (LC-MS/MS) have been developed and validated for assessing the exposure of greenhouse workers to acetamiprid. Both ambient (potential inhalation and dermal exposure) and internal dose (biological monitoring of urine samples) measurements were carried out. Potential inhalation exposure was assessed using Chromosorb 102 cartridges connected to air personal samplers. Potential dermal exposure was estimated by using whole body dosimetry. The measurement of actual exposure was done by analyzing the parent compound in urine samples of the applicators, after a solid-phase extraction (SPE) step. The methods showed a good accuracy (72-92%), precision (2-13%) and lower limits (few microg l(-1)). The validated approaches have been applied to assess potential and actual exposure of agricultural workers spraying acetamiprid in greenhouses. The results shown the need to wear personal protective equipment (suits) in order to reduce the absorbed dose of acetamiprid.     

Improved method for identifying and quantifying olive oil phenolic compounds and their metabolites in human plasma by microelution solid-phase extraction plate and liquid chromatography–tandem mass spectrometry

Two methods based on solid-phase extraction (SPE) using traditional cartridges and microelution SPE plates (μSPE) as the sample pre-treatment, and an improved liquid chromatography coupled to tandem mass spectrometry (UPLC–MS/MS) were developed and compared to determine the phenolic compounds in virgin oil olive from plasma samples. The phenolic compounds studied were hydroxytyrosol, tyrosol, homovanillic acid, p-coumaric acid, 3,4-DHPEA-EDA, p-HPEA-EDA, luteolin, apigenin, pinoresinol and acetoxypinoresinol. Good recoveries were obtained in both methods, and the LOQs and LODs were similar, in the range of low μM. The advantage of μSPE, in comparison with SPE cartridges, was the lack of the evaporation step to pre-concentrate the analytes. The μSPE-UPLC–ESI-MS/MS method developed was then applied to determine the phenolic compounds and their metabolites, in glucuronide, sulphate and methylated forms, in human plasma after the ingestion of virgin olive oil.     

MCX based solid phase extraction combined with liquid chromatography tandem mass spectrometry for the simultaneous determination of 31 endocrine-disrupting compounds in surface water of Shanghai

A novel analytical method employing MCX (mixed-mode cationic exchange) based solid phase extraction (SPE) coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed to detect 31 endocrine-disrupting compounds (EDCs) in surface water samples simultaneously. The target EDCs belong to five classes, including seven estrogens, eight androgens, six progesterones, five adrenocortical hormones and five industrial compounds. In order to simultaneously concentrate the target EDCs and eliminate matrix interferences in the water samples, MCX SPE cartridges were employed for SPE, and then followed by a simple and highly efficient three-step sequential elution procedure. Two electrospray ionization (ESI) detection modes, positive (ESI+) and (ESI-), were optimized for HPLC-MS/MS analysis to obtain the highest sensitivity for all the EDCs. The limits of detection (LODs) were 0.02-1.9 ng L(-1), which are lower than or comparable to these reported in references. Wide linear ranges (LOD-100 ng L(-1) for ESI+ mode, and LOD-200 ng L(-1) for ESI- mode) were obtained with determination coefficients (R(2)) higher than 0.99 for all the compounds. With five internal standards, good recoveries (84.4-103.0%) of all the target compounds were obtained in selected surface water samples. The developed method was successfully applied to investigate the EDCs occurrence in the surface water of Shanghai by analyzing surface water samples from 11 sites. The results showed that nearly all the target compounds (30 in 31) were present in the surface water samples of Shanghai, of which three industrial compounds (4-t-OP, BPA, and BPF) showed the highest concentrations (median concentrations were 11.88-23.50 ng L(-1)), suggesting that industrial compounds were the dominating EDCs in the surface water of Shanghai, and much more attention should be paid on these compounds. Our present research demonstrated that SPE with MCX cartridges combined with HPLC-MS/MS was convenient, efficient and reliable for multiclass analysis of EDCs in surface water.     

Determination of Cloretazine (VNP40101M) and its active metabolite (VNP4090CE) in human plasma by liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS)

A sensitive method for the determination of Cloretazine (VNP40101M) and its metabolite (VNP4090CE) with an internal standard (ISTD) in human plasma was developed using high-performance liquid chromatographic separation with tandem mass spectrometric detection. Acidified plasma samples (500 microL) were prepared using solid phase extraction (SPE) columns, and 25 microL of the reconstituted sample was injected onto an Ascentis C18 HPLC column (3 microm, 5 cmx2.1 mm) with an isocratic mobile phase. Analytes were detected with an API-3000 LC-MS/MS System at unit (Q1) and low (Q3) resolution in negative multiple reaction monitoring mode: m/z 249.0 (precursor ion) to m/z 114.9 (product ion) for both Cloretazine (at 3.64 min) and VNP4090CE (at 2.91 min), and m/z 253.0 (precursor ion) to m/z 116.9 (product ion) for the ISTD. The mean recovery for Cloretazine (VNP40101M) and its metabolite (VNP4090CE) was greater than 87% with a lower limit of quantification of 1.0 ng/mL for Cloretazine (S/N=9.7, CV相似文献   

Determination of a novel gamma-secretase inhibitor in human plasma and cerebrospinal fluid using automated 96 well solid phase extraction and liquid chromatography/tandem mass spectrometry

A method for determination of a gamma-secretase inhibitor, cis-3-[4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]propanoic acid (A), in human plasma and cerebrospinal fluid (CSF) has been developed to support the clinical investigation of compound A for its potential treatment of Alzheimer's disease. The method is based on HPLC with atmospheric pressure chemical ionization tandem mass spectrometric detection (APCI-MS/MS) in the negative ionization mode using a heated nebulizer interface. The addition of phosphoric acid at the ratio of 10-30microL per milliliter of human plasma or CSF was required during clinical sample collection to stabilize an acylglucuronide metabolite (C), which was potentially present in human plasma and CSF. Tween 20 (10% solution) was added at the ratio of 20microL per milliliter of CSF during CSF sample collection to prevent the loss of compound A during the storage of clinical samples. The compound A and its analog internal standard (B) in treated plasma or CSF were isolated from human plasma or CSF using solid phase extraction (SPE) in the 96 well format. The isolated analyte and internal standard were chromatographed on a Phenomenex Synergi Polar RP analytical column (50mmx3.0mm, 4microm), using a mobile phase consisting of 60/40 (v/v, %) acetonitrile/water at a flow-rate of 0.5mL/min. Tandem mass spectrometric detection was performed using a Sciex API 3000 tandem mass spectrometer operated in the multiple reaction monitoring (MRM) mode using precursor to product ion transitions of 441-->175 for A and 469-->175 for B, respectively. The assays were validated over the concentration range of 0.5-200ng/mL for human plasma and CSF. Replicate analyses (n=5) of spiked standards for both assays yielded a linear response with coefficients of variation less than 7% and accuracy within 5% of the nominal concentrations. In addition, the assays were automated to improve sample throughput by utilizing a Packard Multi PROBEII automated liquid handling system and a Tom-Tec Quadra 96 system. Numerous clinical studies have been supported using these assays.     

Enantiomeric determination of amlodipine in human plasma by liquid chromatography coupled to tandem mass spectrometry

A sensitive method for the separation and determination of amlodipine enantiomers in plasma has been developed based on solid-phase extraction (SPE) with disposable extraction cartridges (DECs) in combination with chiral liquid chromatography (LC). The SPE technique is used to isolate the drug from the biological matrix and to prepare a cleaner sample before injection and analysis by HPLC coupled to mass spectrometry. The DEC is filled with ethyl silica (50 mg) and is first conditioned with a 2.5% ammonia in methanol solution and then with ammonium acetate buffer. A 1.0-ml volume of plasma is then applied on the DEC. The washing step is first performed with ammonium acetate buffer and secondly with a mixture of water and methanol (65:35, v/v), while the final elution step is obtained by dispensing methanol containing 2.5% of ammonia. The eluate is then collected and evaporated to dryness before being dissolved in the LC mobile phase and injected into the LC system. The stereoselective analysis of amlodipine is achieved on a Chiral AGP column containing alpha(1)-acid glycoprotein as chiral selector by using a mobile phase consisting of a 10-mM acetate buffer (pH 4.5) and 1-propanol (99:1, v/v). The LC system is coupled to tandem mass spectrometry with an APCI interface in the positive-ion mode. The chromatographed analytes are detected in the selected reaction monitoring mode (SRM). The MS/MS ion transitions monitored are 409 to 238 for amlodipine, and 260 to 116 for S-(-)-propranolol used as internal standard (IS). The method was validated considering different parameters, such as linearity, precision and accuracy. The limit of quantitation was found to be 0.1 ng/ml for each amlodipine enantiomer.     

Control of matrix effects in the analysis of urinary F2-isoprostanes using novel multidimensional solid-phase extraction and LC-MS/MS

F(2)-isoprostanes (F(2)-iPs), established markers of oxidative stress, exist as four sets of regioisomers. Simultaneous and specific determination of F(2)-iPs can be achieved by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We developed novel methods for urine sample preparation and HPLC to control matrix-related ion suppression effects in the LC-MS/MS analysis of F(2)-iPs. A selective solid-phase extraction (SPE) wash protocol was developed with an Oasis HLB (hydrophilic-lipophilic balance) SPE cartridge using an elution profile of [(3)H]8-iso-prostaglandin (PG)F(2alpha) (iPF(2alpha)-III) when the methanol concentration was increased under acidic, neutral, and base wash conditions. A multidimensional (MD)-SPE method that incorporated size exclusion chromatography [corrected] reverse-phase chromatography, and normal-phase chromatography was developed using an Oasis HLB SPE cartridge and an HLB microElution SPE plate. Average extraction recoveries of the deuterated internal standards of iPF(2alpha)-III and iPF(2alpha)-VI were 62 +/- 8% and 60 +/- 10%. A buffer-free HPLC method for the separation of F(2)-iP isomers was developed on base-deactivated C8 columns. Average matrix effects for iPF(2alpha)-III and iPF(2alpha)-VI were 95 +/- 6% and 103 +/- 5%. The clean extraction of urine F(2)-iPs using MD-SPE and the separation of F(2)-iP isomers using a novel HPLC method did not cause apparent ion suppression in the analysis of iPF(2alpha)-III and iPF(2alpha)-VI using LC-MS/MS. These findings should be useful for establishing a routine LC-MS/MS method for the analysis of F(2)-iPs.     

Analytical methods applied to the determination of heterocyclic aromatic amines in foods

Analytical aspects concerning the heterocyclic aromatic amines (HAAs) determination in foods are reviewed. Sample pre-treatment procedures such as liquid-liquid extraction (LLE), supercritical fluid extraction, solid-phase extraction (SPE), solid-phase microextraction (SPME), and the mainly used LLE-SPE tandem extraction are discussed. The analytical methods used for the identification and quantification are HPLC, HPLC combined with single or tandem MS detection (HPLC-MS, HPLC-MS/MS), GC-MS and capillary electrophoresis. Advantages and figures of merit for each technique are discussed.     

Solid-phase extraction on Styrosorb cartridges as a sample pretreatment method in the stereoselective analysis of propranolol in human serum

Sample pretreatment using solid-phase extraction (SPE) on cartridges filled with small-particle Styrosorb porous polystyrene-based sorbent has been used in the analysis of propranolol enantiomers in human serum by high-performance liquid chromatography (HPLC) with fluorescent detection. SPE on Sep-Pak C₁₈ cartridges was used as a reference pretreatment method. The propranolol content of the samples was determined by achiral normal-phase HPLC and the enantiomeric ratio of propranolol (S/R) was then determined by chiral HPLC on a column with silica-bonded cellulose-tris(3,5-dimethylphenyl carbamate). Recoveries of propranolol from serum using SPE on Styrosorb and C₁₈ phases were 97±5% and 96±5%, respectively. Detection and quantification limits for propranolol enantiomers were 4 and 7 ng/ml, respectively.     

Semi-automated solid-phase extraction procedure for the high-performance liquid chromatographic determination of alinastine in biological fluids

A solid-phase extraction (SPE) method for sample clean-up followed by a reversed-phase HPLC procedure for the assay of alinastina (pINN) in biological fluids is reported. The effects of the sample pH, composition of the washing and elution solvents and the nature of the SPE cartridge on recovery were evaluated. The selectivity of SPE was examined using spiked rat urine and plasma samples and the CH and PH cartridges gave rise to the cleanest extracts. The recoveries obtained in spiked rat urine and plasma samples were 91.2±2.7 and 99.9±2.8%, respectively. The proposed SPE method coupled off-line with a reserved-phase HPLC system with fluorimetric detection was applied to the quantitation of alinastine in real rat urine samples. The analytical method was also applied and validated for the determination of alinastine in dog plasma. The recovery from spiked dog plasma samples using the PH cartridge was around 65%. The within-day and between-day precisions were 7 and 12%, respectively. The detection and quantitation limits in dog plasma were 0.024 and 0.078 μg/ml, respectively.     

Determination of 5-S-cysteinyldopa in plasma and urine using a fully automated solid-phase extraction–high-performance liquid chromatographic method for an improvement of specificity and sensitivity of this prognostic marker of malignant melanoma

5-S-Cysteinyldopa (5-SCD) in plasma and urine was determined by means of a newly developed method. This method incorporates optimized conditions for blood collection and storage, as well as a new extraction and separation technique, required for the strong oxidation and light sensitive 5-SCD. The new aspects of the method are the following: immediate centrifugation and freezing of the samples after blood collection, fully automatical solid-phase extraction (SPE) with phenylboronic acid (PBA) cartridges and immediate HPLC injection of the eluate, nearly complete exclusion of light and air–oxygen during extraction, constant sample cooling, use of the more suitable internal standard 5-S- -cysteinyldopa and easy, sensitive and selective HPLC conditions (RP18-column with isocratic separation and electrochemical detection). The method has a linear range from 0.25 to 50 μg l⁻¹ and 25 to 5000 μg l⁻¹ for plasma and urine samples, respectively, a limit of detection of 0.17 μg l⁻¹, intra-assay variabilities from 1.7 to 3.6%, inter-assay variabilities from 4.0 to 18.3% and an average relative recovery of 103.5% for plasma and 105.4% for urine samples. In our study the measured 5-SCD concentrations of patients with melanomas at various stages correlated better with their clinical pictures than described in literature up to date. The results were obtained in comparison to patients with other skin tumors and in comparison to healthy control persons.     

High-performance liquid chromatographic determination of tiapride and its phase I metabolite in blood plasma using tandem UV photodiode-array and fluorescence detection

New bioanalytical SPE-HPLC-PDA-FL method for the determination of the neuroleptic drug tiapride and its N-desethyl metabolite was developed, validated and applied to xenobiochemical and pharmacokinetic studies in humans and animals. The sample preparation process involved solid-phase extraction of diluted plasma spiked with sulpiride (an internal standard) using SPE cartridges DSC-PH Supelco, USA. Chromatographic separation of the extracts was performed on a Discovery HS F5 250 mm × 4 mm (Supelco) column containing pentafluorophenylpropylsilyl silica gel. Mobile phase (acetonitrile-0.01 M phosphate buffer pH=3, flow rate 1 ml min(-1)) in the gradient mode was employed in the HPLC analysis. Tandem UV photodiode-array→fluorescence detection was used for the determination of the analytes. Low concentrations of tiapride and N-desethyl tiapride were determined using a more selective fluorescence detector (λ(exc.)/λ(emiss.)=232 nm/334 nm), high concentrations (500-6000 pmol ml(-1)) using a UV PDA detector at 212 nm with a linear response. Each HPLC run lasted 15 min. Lower limits of quantification (LLOQ) for tiapride (N-desethyl tiapride) were found to be 8.24 pmol ml(-1) (10.11 pmol ml(-1)). The recoveries of tiapride ranged from 89.3 to 94.3%, 81.7 to 86.8% for internal standard sulpiride and 90.9 to 91.8% for N-desethyl tiapride.     

Simultaneous determination of glycyrrhizin, a marker component in radix Glycyrrhizae, and its major metabolite glycyrrhetic acid in human plasma by LC-MS/MS

Glycyrrhizin (GLY) which has been widely used in traditional Chinese medicinal preparation possesses various pharmacological effects. In order to investigate the pharmacokinetic behavior of GLY in human after oral administration of GLY or licorice root, a liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of GLY and its major metabolite glycyrrhetic acid (GA) in human plasma. The method involved a solid phase extraction of GLY, GA, and alpha-hederin, the internal standard (IS), from plasma with Waters Oasis MCX solid phase extraction (SPE) cartridges (30 mg) and a detection using a Micromass Quattro LC liquid chromatography/tandem mass spectrometry system with electrospray ionization source in positive ion mode. Separation of the analytes was achieved within 5min on a SepaxHP CN analytical column with a mobile phase of acetonitrile:water (50:50, v:v) containing 0.1% formic acid and 5mM ammonium acetate. Multiple reaction monitoring (MRM) was utilized for the detection monitoring 823--> 453 for GLY, 471--> 177 for GA and 752--> 456 for IS. The LC-MS/MS method was validated for specificity, sensitivity, accuracy, precision, and calibration function. The assay had a calibration range from 10 to 10,000 ng/mL and a lower limit of quantification of 10 ng/mL for both GLY and GA when 0.2 mL plasma was used for extraction. The percent coefficient of variation for accuracy and precision (inter-run and intra-run) for this method was less than 11.0% with a %Nominal ranging from 87.6 to 106.4% for GLY and 93.7 to 107.8% for GA. Stability of the analytes over sample processing (freeze/thaw, bench-top and long-term storage) and in the extracted samples was also tested and established.