Simultaneous determination of a novel M3 muscarinic receptor antagonist and its active 5-OH metabolite in human plasma using liquid chromatography/tandem mass spectrometry

A sensitive, specific, and robust liquid chromatography (LC)/mass spectrometry (MS)/MS method has been developed and validated for a novel M(3) muscarinic receptor antagonist (I) and its active 5-OH metabolite (II) in human plasma. The assay involves a two-step liquid-liquid extraction of the compounds from human plasma, high performance liquid chromatography (HPLC) separation, and MS/MS for the detection of the analytes. The method provides a linear response from a quantitation limit of 0.05-20 ng/ml for I and 0.1-20 ng/ml for II using 1 ml of plasma. The mean absolute recovery was 85.4% for I and 80.8% for II, respectively. The intra-assay accuracy of I and II averaged from 95.0 to 105.3% with coefficient of variation (CV) values 相似文献   

LC/MS/MS plasma assay for the peptidomimetic VLA4 antagonist I and its major active metabolite II: for treatment of asthma by inhalation.

In vitro and in animals, I is a potent and specific peptidomimetic for the potential treatment of airway inflammation in the pathogenesis of asthma. Preclinical studies indicated extensive conversion of I to an active metabolite II, and thus, a very sensitive assay for I and II was needed to support an inhalation ascending-dose study in man. The LC/MS/MS plasma/urine assay method (1.0 ml of sample) involves the following: liquid-liquid extraction of acidified plasma into pentane-ethyl acetate (90:10 v/v); evaporation of the organic extract, reconstitution into methanol; addition of water to the methanolic extract and freezing. After thawing, the extract is centrifuged and the clear supernatant injected for chromatography. Extract is chromatographed on a YMC ODS-AM column (50 x 2.0 mm). For detection, a Sciex 365 LC/MS/MS with an electrospray inlet and used in the positive ion, multiple reaction monitoring mode was used to monitor precursor-->fragment ions of m/z 709-->594 for I and m/z 513-->380 for II. The plasma assay was linear over the concentration range of 0.1-100 ng/ml in plasma for I and II. Accuracy and precision for I ranged from 97.9 to 102.1% of nominal with a 0.84-10.65% CV; similarly for II, 98.0-101.7% and 1.39-9.28% CV, respectively. Extraction recovery averaged 63.7% for I and 64.9% for II. This general assay methodology may be applied to assay small acidic peptides and peptidomimetics from biological fluids by LC/MS/MS.     

Sensitive determination of nefopam and its metabolite desmethyl-nefopam in human biological fluids by HPLC

Nefopam (NEF) and desmethyl-nefopam (DMN) were assayed simultaneously in plasma, globule and urine samples using imipramine as internal standard. A liquid-liquid extraction procedure was coupled with a reverse phase high-performance liquid chromatography system. This system requires a mobile phase containing buffer (15 mM KH(2)PO(4) with 5 mM octane sulfonic acid: pH 3.7) and acetonitrile (77:33, v/v) through (flow rate=1.5 ml/min) a C(18) Symmetry column (150x4.6 I.D., 5 micrometer particle size: Waters) and a UV detector set at 210 nm. Internal standard was added to 1 ml of plasma or globule sample or 0.5 ml of urine sample, prior to the extraction under alkaline ambiance with n-hexane. The limits of quantification were 1 and 2 ng/ml for both molecules in plasma and globule, respectively; 5 and 10 ng/ml for NEF and DMN in urine, respectively. The method proved to be accurate and precise: the relative error at three concentrations ranged from -13.0 to +12.3% of the nominal concentration for all molecule and biological fluid; the within-day and between-day precision (relative standard deviation %) ranged from 1.0 to 10.1% for all the molecules and biological fluids. The method was linear between 1 and 60 ng/ml for both molecules in the plasma; 2 and 25 ng/ml for both molecules in the globule; 25 and 250 ng/ml for NEF and 50 and 500 ng/ml for DMN in the urine: correlation coefficients of calibration curves (determined by least-squares regression) of each molecule were higher than 0.992 whatever the biological fluid and during the pre-study and in-study validations. This method was successfully applied to a bio-availability study of NEF in healthy subjects.     

Quantitative determination of a novel insulin sensitizer and its para-hydroxylated metabolite in human plasma by LC-MS/MS

I, 5-[3-[3-(4-phenoxy-2-propylphenoxy)-propoxy]-phenyl]-2,4-thiazolidinedione sodium salt, is a dual alpha/gamma peroxisome proliferator-activated receptor (PPAR) agonist for potential use in diabetic patients. The compound has a para-hydroxylated metabolite, II, which has also been shown to exhibit PPAR activity. An LC-MS/MS method for the simultaneous determination of I and its active metabolite (II) in human plasma has been successfully developed. The method consists of treating 0.5 ml plasma with ammonium acetate (pH 9.6; 50mM) and extracting I, II and internal standard (III, Fig. 2) with 5 ml ethyl acetate. The ethyl acetate is evaporated and the samples are reconstituted in 0.1 ml acetonitrile:0.1% formic acid (65:35, v/v). The entire extraction procedure, as well as sample collection, was performed in glass tubes and vials to overcome the analytes adherence to polypropylene. A linear HPLC gradient was used to separate the analyte, metabolite, internal standard, and other interfering, non-quantitated metabolites. Detection was by negative ionization MS/MS on a turbo ionspray probe. Precursor-->product ion combinations were monitored in multiple reaction monitoring (MRM) mode. The linear range is 0.05-20 ng/ml for I and 0.1-20 ng/ml for II. Recoveries were 59.4, 90.1 and 56.8% for I, II and III, respectively. Intraday variation using this method was <==7.0% for I and <==9.2% for II. The method exhibits good linearity and reproducibility for each analyte and good sensitivity, selectivity and robustness when used for the analysis of clinical samples.     

A validated SPE-LC-MS/MS assay for Eplerenone and its hydrolyzed metabolite in human urine

An automated LC-MS/MS assay was validated to quantitate the first selective aldosterone blocker Eplerenone (I) and its hydrolyzed metabolite (II) in human urine. After the addition of the stable isotope labeled internal standards, human urine samples were extracted on a C(18) solid phase extraction (SPE) cartridge using a Zymark RapidTrace automation system. The extraction eluates were diluted with 20 mM ammonium acetate aqueous solution and directly injected onto the LC-MS/MS system. The chromatographic separation was performed on a reverse phase Zorbax XDB-C(8) HPLC column (2.1 x 50 mm, 5 microm) with a mobile phase of acetonitrile:water (40:60, v/v) containing 10 mM ammonium acetate (pH 7.4). I and II were ionized using positive and negative ionization mass spectrometry, respectively, to achieve the best sensitivity. The ionization polarity was switched during the run at approximately 2.5 min after the injection. Multiple reaction monitoring (MRM) with a tandem mass spectrometer was used to detect the analytes. The precursor to product ion transitions of m/z 415-->163 and m/z 431-->337 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 50-10000 ng/ml of urine for both of I and II. The lower limit of quantitation (LLOQ) was 50 ng/ml for I and II. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. Sample analysis time for each injection was 5 min; a throughput of 100 human urine standards and samples per run was achieved.     

Extended application of an LC-MS/MS method for the analysis of vesnarinone and its metabolites in human urine and dialysate fluid

Vesnarinone, a positive inotropic drug developed for congestive heart failure, and its metabolites (OPC-8230, OPC-18136, OPC-18137) were analyzed in human dialysate and urine (plus an additional metabolite: OPC-18692 in urine) samples using a modification to a previously published LC-MS/MS assay for the analysis of human plasma and urine samples. OPC-8192, a structural analogue of vesnarinone, was used as the internal standard. The analytes of interest were extracted from human dialysate or urine by a solid phase extraction method using a pre-conditioned C-18 extraction column. The analytes were then resolved by a 7 min gradient elution on a reverse phase high performance liquid chromatographic column. Vesnarinone and metabolites were detected on a PE/Sciex API III+Biomolecular Mass analyzer in MS/MS mode using a Turbo IonSpray interface. The linear range of quantitation in dialysate was 2.00-100.00 ng/ml for vesnarinone and 0.50-25.00 ng/ml for each metabolite. In urine, the linear range was of 0.50-25.00 microg/ml for vesnarinone and 0.10-5.00 microg/ml for the metabolites. This method was used to support the analysis of urine and dialysate samples from renally impaired patients who are on vesnarinone treatment.     

LC/MS/MS quantitation of an anti-cancer drug in human plasma using a solid-phase extraction workstation: application to population pharmacokinetics

A liquid chromatographic/mass spectrometric (LC/MS/MS) method to quantitate an anti-cancer drug in human plasma was validated. The method has proven suitable for routine quantitation of the experimental anti-cancer compound at concentrations from 1 to 400 ng/ml. Retention times of the compound and internal standard (compounds I and II, respectively) were 1.8 and 2.1 min, respectively. No interfering endogenous peaks were observed throughout the validation process. Precision estimates for this approach were typically less than 5% relative standard deviation (RSD) across the calibration range. Other validation parameters studied included specificity, system reproducibility, limit of quantitation, accuracy, linear range, and stability of the compound and internal standard in plasma and injection solvent. This method was used to quantify drug for population pharmacokinetic studies.     

Development and validation of an automated SPE-LC-MS/MS assay for valdecoxib and its hydroxylated metabolite in human plasma

A sensitive and specific liquid chromatography-tandem mass spectrometry assay was developed to quantitate valdecoxib (I) and its hydroxylated metabolite (II) in human plasma. The analytes (I and II) and a structurally analogue internal standard (IS) were extracted on a C(18) solid phase extraction (SPE) cartridge using a Zymark RapidTrace automation system. The chromatographic separation was performed on a narrow-bore reverse phase Zorbax XDB-C(8) HPLC column with a mobile phase of acetonitrile:water (50:50, v/v) containing 10 mM ammonium acetate. The analytes were ionized using negative electrospray mass spectrometry, then detected by multiple reaction monitoring (MRM) with a tandem mass spectrometer. The precursor to product ion transitions of m/z 313-->118 and m/z 329-->196 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.5-200 ng/ml of I and II in human plasma with absolute recoveries from plasma at 91 and 86%, respectively. The lower limit of quantitation was 0.5 ng/ml for I and II. Acceptable precision and accuracy were obtained for concentrations over the calibration curve ranges (0.5-200 ng/ml). Sample analysis time for each injection was 5 min, a throughput of 70 human plasma standards and samples per run was achieved. The assay has been successfully used to analyze human plasma samples to support clinical phase I and II studies.     

Sensitive determination of erdosteine in human plasma by use of automated 96-well solid-phase extraction and LC-MS/MS

A sensitive and selective method for quantitation of erdosteine in human plasma was established by use of 96-well solid-phase extraction (SPE) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS). Plasma samples were transferred into 96-well OASIS HLB extraction plate using an automated sample handling system and the drugs were eluted with methanol. The eluents were then evaporated and reconstituted with mobile phase. All sample transfer and SPE was automated through the application of both the Perkin-Elmer MultiPROBE II HT and TOMTEC Quadra 96 workstation. Compounds were separated on a C18 column with 1 mM ammonium acetate-acetonitrile (80:20, pH 3.2), as mobile phase at a flow rate of 0.3 ml/min. The limit of quantitation (LOQ) was 0.2 ng/ml, using a sample volume of 0.2 ml for the analysis. The reproducibility of the method was evaluated by analyzing three at 14 quality control (QC) levels over the nominal concentration range from 0.2 to 5000 ng/ml. The intraday accuracy was found to range from 99.6 to 105.0% with precision (% RSD) of less than 4.76% at five QC levels. The interday accuracy was found to range from 95.0 to 100.5% with precision of less than 5.26% at five QC levels. Erdosteine produced a protonated precursor ion ([M+H]+) at m/z 250, and a corresponding product ion at m/z 204. Internal standard (letosteine) produced a protonated precursor ion ([M+H]+) at m/z 280 and a corresponding product ion at m/z 160. The high sample throughput of the method has been successfully applied to a pharmacokinetic study of erdosteine in human plasma.     

Development and validation of a sensitive LC-MS/MS method for the determination of adefovir in human serum and urine

A sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of adefovir (PMEA) in human serum and urine. The analyte was separated on a Diamonsil C(18) column (250 mm x 4.6 mm i.d., 5 microm particle size) by isocratic elution with methanol-water-formic acid (20:80:0.1, v/v/v) at a flow rate of 0.6 ml/min, and analyzed by mass spectrometry in multiple reaction-monitoring mode. The precursor-to-product ion transitions of m/z 274-->162 and m/z 226-->135 were used to measure and quantify the analyte and internal standard (I.S.), respectively. The weighted (1/x(2)) calibration curve was linear over serum concentration range 1.25-160.00 ng/ml and urine concentration range 0.05-8.00 microg/ml, with a correlation coefficient (r) of 0.9992 and 0.9978, respectively. The lower limit of quantification in human serum was 1.25 ng/ml. The inter- and intra-day precisions (R.S.D.%) in both serum and urine were lower than 8.64%, the mean method accuracies and recoveries from spiked serum samples at three concentrations ranged from 96.3 to 102.0% and 56.5 to 59.3%, respectively. The serum extract was stable when stored for 24h. The developed method was successfully applied to determine PMEA in human serum and urine, and proved suitable to clinical pharmacokinetic study.     

Simultaneous determination of losartan and EXP3174 in human plasma and urine utilizing liquid chromatography/tandem mass spectrometry

Losartan is an orally active angiotensin II receptor antagonist indicated for the treatment of hypertension. EXP3174 is an active metabolite, which contributes to the overall activity of losartan. Analytical methods for the simultaneous determination of losartan and its active metabolite EXP3174 in human plasma and urine with limited plasma sample size have been developed and validated to support a pediatric clinical program. In both methods, analytes are extracted from the matrixes by liquid-liquid extraction and separated using reverse phase high-performance liquid chromatography (HPLC). A tandem mass spectrometer (MS/MS) with a Turbo ionspray (TIS) interface in multiple-reaction-monitoring (MRM) mode is used for detection of the analytes in both methods. The plasma method has a lower limit of quantitation (LOQ) of 1 ng/ml with a linearity range of 1-500 ng/ml for losartan and EXP3174 using 100 microl of plasma. For the urine method, the LOQ for both losartan and EXP3174 is 2 ng/ml using 0.5 ml of urine, and the linearity range for both analytes is 2-1000 ng/ml. Validation procedures have proven that both methods are robust, accurate, and reproducible. Both methods have been used to assay clinical samples and provided satisfactory results.     

Determination of a beta(3)-agonist in human plasma by LC/MS/MS with semi-automated 48-well diatomaceous earth plate

Methods for the determination of a beta(3)-agonist (A) in human plasma were developed and compared based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection using a turbo ion spray (TIS) interface. Drug and internal standard were isolated from plasma by three sample preparation methods, liquid-liquid extraction, Chem Elut cartridges and 48-well diatomaceous earth plates, that successively improved sample throughput for LC/MS/MS. MS/MS detection was performed on a PE Sciex API 365 tandem mass spectrometer operated in positive ion mode and using multiple reaction monitoring (MRM). The precursor/product ion combinations of m/z 625/607 and 653/515 were used to quantify A and internal standard, respectively, after chromatographic separation of the analytes. Using liquid-liquid extraction and Chem Elut cartridges, the assay concentration range was 0.5-100 ng/ml. Using diatomaceous earth plates, the concentration range of the assay was extended to 0.5-200 ng/ml. For all three assays, the statistics for precision and accuracy is comparable. The assay accuracy ranged from 91-107% and intraday precision as measured by the coefficient of variation (CV) ranged 2-10%. The sample throughput was tripled when the diatomaceous earth plate method was compared with the original liquid-liquid extraction method.     

Pharmacokinetics of amoxicillin in human urine using online coupled capillary electrophoresis with electrogenerated chemiluminescence detection

A novel and sensitive method for the determination of amoxicillin (AM) in human urine has been established using capillary electrophoresis (CE) coupled with electrochemiluminescence (ECL) detection, based on the ECL enhancement of Tris(2,2'-bipyridyl) ruthenium(II) with AM. The effects of several factors such as the detection potential, the concentration and the pH of phosphate buffer, the electrokinetic voltage and the injection time were investigated. Under the optimal conditions, the linear concentration of AM ranged from 1.0 ng/ml to 8.0 microg/ml (with a correlation coefficient of 0.9999). The limit of detection was 0.31 ng/ml. The mean recovery was 95.77% with relative standard deviations of no larger than 2.2%. This method is quick (the total run time within 6 min). This method has been successfully applied to a pharmacokinetic study in human urine after oral administration of AM.     

Simultaneous determination of a novel thrombin inhibitor and its two metabolites in human plasma by liquid chromatography/tandem mass spectrometry

I, 3-(2-phenethylamino)-6-methyl-1-(2-amino-6-methyl-5-methylene-c arboxamidomethylpyridinyl)-pyrazinone dihydrochloride monohydrate, is a potent, orally active thrombin inhibitor. The compound also has two metabolites which have been shown to have thrombin inhibitory activity: benzylic alcohol M3 metabolite (II) and hydroxymethylpyrazinone M7 metabolite (III). A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of I and its two metabolites in human plasma has successfully been developed. The method consists of treating 0.5 ml plasma with 2 N NaOH and extracting I, II, III and internal standard (IV) with ethyl acetate:methyl-t-butyl ether (1:3, v/v). The analytes were then back-extracted into 2% formic acid. The analytes were chromatographed by high performance liquid chromatography (HPLC) and detected by MS/MS. Positive ionization was used on a heated nebulizer probe monitoring precursor --> product ion combinations in multiple reaction monitoring mode. The linear range is 0.5-1000 nM for I and III and 0.75-1000 nM for II. Recoveries were 88, 74, 78 and 102.1% for I, II and III and the internal standard, respectively in human plasma. Intraday variation using this method was < or = 7.9% for I, < or = 9.0%, for II and < or = 9.5% for III across the standard curve range. This method exhibits good linearity and reproducibility for the three analytes.     

A selective and rapid method for the quantification of captopril in human plasma using liquid chromatography/selected reaction monitoring mass spectrometry

A specific hyphenated high performance liquid chromatography–mass spectrometric (LC–MS/MS) assay was developed for the determination of captopril in plasma. The drug was extracted from plasma using liquid–liquid extraction with a mixture of diethylether:dichloromethane. After the addition of the internal standard, samples were applied to a prepacked C₈ Waters Symmetry column. The ion trap MS/MS detector was equipped with electrospray ionization (ESI) source operating in the positive ion mode. Drug determination was accomplished monitoring captopril at molecular ion m/z 218 and MS/MS (daughter) at m/z 171.6. The method was applied to captopril determination in human plasma after the administration of captopril 50 mg tablets to healthy volunteers who have participated in a pharmacokinetic study.

The method was proved to be specific and precise by testing six different plasma batches. Linearity was established for the range of concentrations 25–3000 ng/ml with a regression factor of 0.9995. Intra-day accuracy ranged from 90.16 to 96.18%, while the intra-day precision ranged from 2.60 to 9.66% at the concentrations of 75, 1440 and 2500 ng/ml. Inter-day precision of the method ranged from 5.04 to 10.10%. This validated method of analysis was successfully applied to human plasma analyses after the administration of a single dose of 50 mg captopril tablets to healthy volunteers.     

Quantification of sofalcone in human plasma and urine by high performance liquid chromatography-mass spectrometry

Sofalcone, isolated from the root of the Chinese medicinal plant Sophora subprostrata, is well known to be a mucosal protective agent for gastritis and peptic ulcer treatment. Although the LC-MS/MS and HPLC-DAD methods for assay of plasma concentration of sofalcone were reported before for the pharmacokinetic study, they were either too complicated or not sensitive enough for current pharmacokinetic study. In addition, no urinary assay method or pharmacokinetic information was available. Thus an improved high performance liquid chromatography-mass spectrometric method employing negative electrospray ionization was developed for the determination of sofalcone concentration in human plasma and urine sample. A liquid-liquid extraction method was utilized to extract sofalcone together with the indometacin (internal standards) from 0.5ml of human plasma or urine samples. Multiple reaction monitoring was used for quantification by monitoring the transition of m/z from 449.5 to 313.1 for sofalcone and 356.9 to 313.0 for IS. The validation of the method regarding to specificity, sensitivity, linearity, reproducibility, accuracy and stability was evaluated. The lower limit of quantification (LLOQ) of the developed assay method for sofalcone was 0.5ng/ml and the linear calibration curve was acquired with R(2)>0.99 between 0.5 and 500ng/ml for both plasma and urine samples. The intra- and inter-day variations of the current assay were evaluated with the relative standard deviation (RSD) within 13.77% at low concentration of quality control samples (QCs) and 8.71% for other QCs, whereas the mean accuracy ranged from 96.21 to 107.33%. The samples were found to be stable under the storage conditions at least for a month and other experimental conditions. This validated method was then utilized to test sofalcone concentration in clinical samples. Based on these data, the pharmacokinetic behavior of sofalcone in plasma as well as urine was described. As a conclusion, the present method proved to be a rapid and sensitive analytical tool for sofalcone in human plasma or urine samples and has been successfully applied to a clinical pharmacokinetic study of in healthy Chinese subjects.     

Quantitative determination of paclitaxel in human plasma using semi-automated liquid-liquid extraction in conjunction with liquid chromatography/tandem mass spectrometry

This paper describes a high-throughput sample preparation procedure combined with LC-MS/MS analysis to measure paclitaxel in human plasma. Paclitaxel and an internal standard were extracted from plasma by a semi-automated robotic method using liquid-liquid extraction. Thereafter compounds were separated on a RP C18 column. Detection was by a PE Sciex API 3000 mass spectrometer equipped with a TurboIonSpray interface. The compounds were detected in positive ion mode using the mass transition m/z 854.6-->286.2 and m/z 831.6-->263.2 for paclitaxel and the internal standard, respectively. The limit of quantitation for paclitaxel was 1 ng/ml with an imprecision of 5.2% following extraction of 0.1 ml of plasma. Linearity was confirmed over the whole calibration range (1-1000 ng/ml) with correlation coefficients higher than 0.99 indicating good fits of the regression models. The inter and intra-day precision was better than 9.5% and the accuracy ranged from 90.3 to 104.4%. The assay was simple, fast, specific and exhibited excellent ruggedness.     

A subnanogram API LC/MS/MS quantitation method for depsipeptide FR901228 and its preclinical pharmacokinetics

A highly sensitive and specific atmospheric pressure ionization (API) liquid chromatographic-tandem mass spectrometric (LC/MS/MS) method for the quantitation of depsipeptide FR901228 (NSC-630176, FR), a naturally occurring antitumor agent, was developed and validated. FR was extracted from human or rat plasma along with the internal standard, t-Boc-Met-Leu-Phe (BMLP) with ethyl acetate. Components in the extract were separated on a 5-microm C8 Spherisorb 50 x 4.6 mm i.d. column by isocratic elution with methanol/acetonitrile/12 mM ammonium acetate (60:10:30, v/v/v). The liquid flow was passed through a presource splitter and 5% of the eluate was introduced into the API source. The components were analyzed in the multiple-reaction monitoring (MRM) mode to enhance specificity. Linear calibration curves were obtained in the range of 0.1-100.0 ng/ml with 0.5 ml human plasma and 0.5-100.0 ng/ml with 0.1 ml rat plasma. The limit of quantitation (LOQ) was 0.1 ng/ml using 0.5 ml human plasma and 0.5 ng/ml using 0.1 ml rat plasma. The overall within-day precision was below 12% in human plasma and below 7% in rat plasma; and the between-day precision was below 10.2% in human plasma and 7.2% in rat plasma. The accuracy at low, medium and high levels ranged from 99.3 to 111.7% in human plasma and 96.2-107.3% in rat plasma. The high sensitivity permitted pharmacokinetic study of FR in the rat at a single i.v. dose as low as 1 mg/kg. At this dose, plasma FR levels declined biexponentially with a mean terminal t(1/2) of 187.7 min (n = 6) and were detectable up to 24 h. After an oral dose at 5 mg/kg, plasma FR levels were highly erratic and yielded a mean bioavailability of 1.6% (n = 6). At a higher oral dose of 50 mg/kg, a mean bioavailability of 10.6% was obtained, both being estimated by a non-crossover method.     

Simultaneous determination of ginsenoside Rb(1) and Rg(1) in human plasma by liquid chromatography-mass spectrometry

A liquid chromatographic-mass spectrometric (LC/MS) method for the simultaneous determination of ginsenoside Rb(1) and Rg(1) in human plasma was developed. The method involved the protein precipitation followed by analysis of ginsenoside Rb(1) and Rg(1) in an Atlantis C(18) column with the gradient elution of acetonitrile and ammonium formate (10mM, pH 3.0) at a flow rate of 0.2 ml/min. The analytes were determined using electrospray negative ionization mass spectrometry in the selected ion monitoring mode. The standard curves for ginsenoside Rb(1) and Rg(1) were linear over the concentration range of 10.0-1000 ng/ml. The lower limit of quantification was 10.0 ng/ml using 100 microl plasma sample. The coefficient of variation of intra- and inter-day assays for ginsenoside Rb(1) and Rg(1) at three quality control levels ranged from 1.0 to 6.8% and 5.4 to 9.8%, respectively. Ginsenoside Rb(1) and Rg(1) were stable in blank human plasma at room temperature for 24h and following three freeze-thaw cycles.