Determination of ciprofloxacin in human plasma using high-performance liquid chromatography coupled with fluorescence detection: application to a population pharmacokinetics study in children with severe malnutrition

Clinical pharmacokinetic studies of ciprofloxacin require accurate and precise measurement of plasma drug concentrations. We describe a rapid, selective and sensitive HPLC method coupled with fluorescence detection for determination of ciprofloxacin in human plasma. Internal standard (IS; sarafloxacin) was added to plasma aliquots (200 μL) prior to protein precipitation with acetonitrile. Ciprofloxacin and IS were eluted on a Synergi Max-RP analytical column (150 mm×4.6 mm i.d., 5 μm particle size) maintained at 40°C. The mobile phase comprised a mixture of aqueous orthophosphoric acid (0.025 M)/methanol/acetonitrile (75/13/12%, v/v/v); the pH was adjusted to 3.0 with triethylamine. A fluorescence detector (excitation/emission wavelength of 278/450 nm) was used. Retention times for ciprofloxacin and IS were approximately 3.6 and 7.0 min, respectively. Calibration curves of ciprofloxacin were linear over the concentration range of 0.02-4 μg/mL, with correlation coefficients (r(2))≥0.998. Intra- and inter-assay relative standard deviations (SD) were <8.0% and accuracy values ranged from 93% to 105% for quality control samples (0.2, 1.8 and 3.6 μg/mL). The mean (SD) extraction recoveries for ciprofloxacin from spiked plasma at 0.08, 1.8 and 3.6 μg/mL were 72.8±12.5% (n=5), 83.5±5.2% and 77.7±2.0%, respectively (n=8 in both cases). The recovery for IS was 94.5±7.9% (n=15). The limits of detection and quantification were 10 ng/mL and 20 ng/mL, respectively. Ciprofloxacin was stable in plasma for at least one month when stored at -15°C to -25°C and -70°C to -90°C. This method was successfully applied to measure plasma ciprofloxacin concentrations in a population pharmacokinetics study of ciprofloxacin in malnourished children.     

Solid-phase extraction and high-performance liquid chromatographic determination of tamoxifen and its major metabolites in plasma

Tamoxifen (TAM) is a triphenylethylene anti-oestrogen, commonly used in the treatment of breast cancer. Patients receiving tamoxifen therapy may experience both de novo and acquired resistance. As one of the mechanisms for this may be extensive peripheral bio-transformation of tamoxifen, there has been considerable interest in the pharmacokinetics and metabolism of tamoxifen. A reversed-phase high-performance liquid chromatography separation has been developed to determine the levels of tamoxifen and its major metabolites in human plasma. The method is highly sensitive (2 ng/ml) and selective for tamoxifen, cis-tamoxifen (CIS), 4-hydroxytamoxifen (4-OH) and desmethyltamoxifen (DMT). A μBondapak C₁₈ 10 μm column (30 cm × 3.9 mm I.D.) was used, with a mobile phase of methanol-1% triethylamine at pH 8 (89:11, v/v). Sample preparation was carried out using a C₂ (500 mg sorbent, 3 ml reservoirs) solid phase extraction method, and extraction efficiencies were approximately 60% for TAM and its metabolites. Accuracy and precision, as determined by spiking plasma samples with a mixture of tamoxifen and its metabolites, ranged from 85–110% (± 5–10%) at 1 μg/ml, 101–118% (± 8–20%) at 0.1 μg/ml and 111–168% (± 43–63%) at 0.01 μg/ml. Results from 59 patients show mean values of 54 ng/ml for 4-OH; 190 ng/ml for DMT; 93 ng/ml for TAM and 30 ng/ml for CIS (detected in three patients only). This methodology can be applied routinely to the determination of TAM and its metabolites in plasma from patients undergoing therapy.     

Analysis of tamoxifen, N-desmethyltamoxifen and 4-hydroxytamoxifen levels in cytosol and KCl-nuclear extracts of breast tumours from tamoxifen treated patients by gas chromatography-mass spectrometry (GC-MS) using selected ion monitoring (SIM)

Tamoxifen, 4-hydroxytamoxifen and desmethyltamoxifen levels were measured in cytosolic and 0.5 M KCl extracted nuclear fractions from a small series of breast tumours from tamoxifen treated patients by gas chromatography-mass spectrometry (GC-MS) using selected ion monitoring (SIM). Tamoxifen and desmethyltamoxifen were the most abundant metabolites. There was a small increment in the relative abundance of 4-hydroxytamoxifen in the nuclear extract over cytosol relative to both tamoxifen and desmethyltamoxifen. Further, there was a selective retention of tamoxifen relative to desmethyltamoxifen in the nuclear extract relative to the cytosol. It is concluded that all three compounds could potentially contribute to estrogen receptor mediated antiestrogenic effects in this target tissue.     

Induction of hepatic drug-metabolising enzymes and tamoxifen metabolite profile in relation to administration route during low-dose treatment in nude rats

Tamoxifen is the most used anticancer drug and is approved for chemoprevention. Little is known about the enzyme inducing properties of low-dose regimens and the influence of route of administration. In this study, nude rats received 5 mg/kg/day of tamoxifen orally or a 50 mg continuous-release pellet subcutaneously. The mRNAs for cytochrome P450-enzymes (CYPs), flavin-containing monooxygenase 1 (FMO1) and phase II drug-metabolising enzymes were quantified by real-time RT-PCR. Tamoxifen and metabolite concentrations were measured using HPLC. We observed a significant increase in CYP3A18 and FMO1 mRNA expression levels in the orally treated animals, whereas the increase in CYP3A2 expression did not reach statistical significance (p = 0.057). No significant induction of enzyme expression was observed in rats that received subcutaneous (S.c.) treatment. After 33 days the serum levels of 4-hydroxytamoxifen (4OHtam), tamoxifen and N-desmethyltamoxifen (NDtam) in orally treated animals were 1.8 ± 0.7, 11.1 ± 3.2 and 11.4 ± 3.8 ng/ml, respectively. In subcutaneously treated animals, tamoxifen and N-desmethyltamoxifen were detected in tissues, but not in serum. These data demonstrate that in contrast to the subcutaneous administration, low-dose oral tamoxifen induced tamoxifen-metabolising enzymes. Furthermore, the different routes of administration resulted in different serum and tissue levels of tamoxifen and metabolites.     

Development and validation of a quantitative assay for the determination of tamoxifen and its five main phase I metabolites in human serum using liquid chromatography coupled with tandem mass spectrometry

A sensitive bioanalytical assay for the quantitative determination of tamoxifen and five of its phase I metabolites (N-desmethyltamoxifen, N-desmethyl-4-hydroxytamoxifen, N-desmethyl-4'-hydroxytamoxifen, 4-hydroxytamoxifen and 4'-hydroxytamoxifen) in serum is described. The method has been fully validated at ranges covering steady-state serum concentrations in patients receiving therapeutic dosages of tamoxifen. The bioanalytical assay is based on reversed phase liquid chromatography coupled with tandem mass spectrometry in the positive ion mode using multiple reaction monitoring for drug (-metabolite) quantification. The sample pretreatment consists of protein precipitation with acetonitrile using only 50 μL of serum. In the past, numerous assays have been developed by other groups for the quantification of tamoxifen and its phase I metabolites. However, the number of metabolites included in these studies is very limited and only very few of these assays have been fully validated. A liquid chromatography tandem mass spectrometry assay for the quantification of tamoxifen and four phase I metabolites in human serum that was previously developed by our group is now explicitly improved and described herein. Time of analysis has been reduced by 50% and sensitivity was increased by a reduction of the lower limit of quantification from 1.0 to 0.2 ng/mL for 4-hydroxytamoxifen and 4'-hydroxytamoxifen. Additionally, two phase I metabolites that have never been quantified in human serum hitherto, namely 4'-hydroxytamoxifen and N-desmethyl-4'-hydroxytamoxifen, were included in this assay. Validation results demonstrate an accurate and precise quantification of tamoxifen, N-desmethyltamoxifen, N-desmethyl-4-hydroxytamoxifen, N-desmethyl-4'-hydroxytamoxifen, 4-hydroxytamoxifen and 4'-hydroxytamoxifen in human serum. The applicability of the assay was demonstrated and it is now successfully used to support clinical studies in which patient-specific dose optimization is performed based on serum concentrations of tamoxifen metabolites.     

Development and validation of a highly rapid and sensitive LC-MS/MS method for determination of SZ-685C, an investigational marine anticancer agent, in rat plasma--application to a pharmacokinetic study in rats

A sensitive and rapid method was developed and validated for the quantitative analysis of the novel anticancer agent SZ-685C in rat plasma using high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) in negative ion mode in order to support the following pre-clinical and clinical studies. SZ-685C and the internal standard (IS, emodin) were extracted from rat plasma by a simple liquid-liquid extraction technique using ethyl acetate as extraction solvent. Chromatographic separation was performed on an Elite Hypersil BDS C18 column (100 mm × 2.1 mm i.d., 3 μm). Elution was carried out using methanol/acetonitrile/2mM ammonium formate (pH 4) (80:15:5 (v/v/v)) at a flow-rate of 0.3 mL/min with a run time of 2.5 min. This assay was linear over a concentration range of 50-10,000 ng/mL with a lower limit of quantification of 50 ng/mL. The intra- and inter-batch precision was less than 15% for all quality control samples at concentrations of 100, 1000 and 7500 ng/mL. These results indicate that the method was efficient with a short run time and acceptable accuracy, precision and sensitivity. This method was successfully applied to explore pharmacokinetics of SZ-685C in rats after oral and intravenous administration of this agent. The absolute bioavailability is about 54.8-66.8% and the t(1/2) is 5.7-9.2h, these results provide basic information for further comprehensive pre-clinical research.     

Chiral assay of omeprazole and metabolites and its application to a pharmacokinetics related to CYP2C19 genotypes

Studies investigating the relationship between CYP2C19 genotype and the stereoselective metabolism of omeprazole have not been reported. In the present study, we developed a simple and sensitive analytical method based on column switching reversed phase high-performance liquid chromatography (HPLC) with UV detection to determine the concentrations of (R)- and (S)-omeprazole and of its principal metabolites, (R)- and (S)-5-hydroxyomeprazole, and the non-chiral, omeprazole sulfone, in human plasma. Sample preparation involved liquid-liquid extraction with diethyl ether:dichloromethane (60:40, v/v) followed by clean-up on a TSK BSA-ODS/S column (5 μm, 10 mm × 4.6mm i.d.) using phosphate buffer:acetonitrile (97:3, v/v, pH 6.4). After column switching, separation was performed on a Shiseido CD-ph chiral column (5 μm, 150 mm × 4.6mm i.d.) using phosphate buffer:methanol (45:55, v/v, pH 5.0) as mobile phase. The limit of quantitation (LOQ) was 5 ng/mL for all analytes with intra- and inter-day precisions (as coefficient of variation) of <9.5% and <9.6%, respectively for all analytes. The present method was successfully applied to a chiral pharmacokinetic study of omeprazole in human volunteers with different CYP2C19 genotypes. The results show that the formation of (R)-5-hydroxyomeprazole gives the best correlation with CYP2C19 genotype.     

Quantification of tamoxifen and three metabolites in plasma by high-performance liquid chromatography with fluorescence detection: application to a clinical trial

A sensitive and reproducible assay employing liquid-liquid extraction and high-performance liquid chromatography with fluorescence detection for the quantification of tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen, and Z-4-hydroxy-N-desmethyltamoxifen in human plasma is described. The compounds and internal standard, propranolol, were separated with a cyano column and a mobile phase of acetonitrile-20 mM potassium phosphate buffer (pH 3; 35:65, v/v) then detected with fluorescence using a modified version of a method originally described by Fried and Wainer [J. Chromatogr. B 655 (1994) 261]. The coefficients of variation for the midpoint of the standard curve for each compound were less than 10%. This method was applied to a pharmacokinetic study of tamoxifen disposition in breast cancer patients.     

Validated liquid chromatography–tandem mass spectrometry method for quantitative determination of dauricine in human plasma and its application to pharmacokinetic study

A highly sensitive and selective LC–MS/MS method was developed and validated for the determination of dauricine in human plasma, using protopine as internal standard (IS). The analyte and IS were extracted by liquid–liquid extraction and analyzed by LC–MS/MS. Chromatographic separation was performed on Agilent TC-C₁₈ column with a mobile phase of methanol–water–glacial acetic acid (60:40:0.8, v/v/v) at a flow rate of 0.7 mL/min. Detection was performed on a triple quadrupole tandem mass spectrum by multiple reaction monitoring (MRM) mode using the electrospray ionization technique in positive mode. The method was linear over the concentration range of 1–200 ng/mL. The lower limit of quantification (LLOQ) was 1 ng/mL in human plasma with acceptable precision and accuracy. The intra- and inter-day precision was less than 5.9% determined from quality control (QC) samples at concentrations of 2.0, 20.0 and 160 ng/mL, and the accuracy was within ±9.9%. This method was successfully applied for the evaluation of pharmacokinetics of dauricine after oral doses of 100, 300 and 600 mg phenolic alkaloids of menispermum dauricum tablet (PAMDT) to 12 Chinese healthy volunteers.     

Interactions of tamoxifen in the chicken

The triphenylethylene antiestrogens are very potent antagonists of estrogen action in the chicken and manifest little agonist activity compared to their action in other species. The estrogen antagonism is most probably mediated by the estrogen receptor, to which tamoxifen binds with a Ki of 2.6 nM. Tamoxifen is readily metabolized by liver to 4-hydroxytamoxifen, which binds the liver nuclear estrogen receptor with a Ki of 0.1 nM. The Kd of the receptor is 0.7 nM. Estrogen receptor concentrations in liver from immature chickens are relatively low both in nuclear and cytosol fractions. Treatment with estradiol results in 10-fold up-regulation of the nuclear levels to give a total receptor concentration of about 2 pmol/g tissue. Tamoxifen can promote this up-regulation to a limited extent, but interpretation of experimental results is compromised by difficulties with exchange assays in the face of the very high binding affinity of 4-hydroxytamoxifen. Tamoxifen also binds with high affinity (Kd 2-4 nM) and distinctive specificity to antiestrogen binding sites (AEBS) present in a wide variety of chicken tissues and in the highest concentration in the liver (800 pmol/g tissue). Liver and serum contain ether-soluble components which can compete for binding of [3H]tamoxifen to the AEBS. The serum AEBS inhibitory activity is chromatographically heterogeneous and is associated with a sterol-like fraction as well as with a fatty-acid-containing fraction. Tamoxifen treatment of cockerels results in dose- and time-dependent decreases in serum free and esterified cholesterol, and in phospholipids and triglycerides. These changes may reflect estrogen-receptor-independent interactions of tamoxifen.     

Rapid and simultaneous determination of nifedipine and dehydronifedipine in human plasma by liquid chromatography-tandem mass spectrometry: Application to a clinical herb-drug interaction study

Nifedipine (NIF), a calcium channel antagonist, is metabolized primarily by cytochrome P450 (CYP3A4) to dehydronifedipine (DNIF). As such, NIF is often used as a probe drug for determining CYP3A4 activity in human studies. A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated to simultaneously determine NIF and DNIF in human plasma using nitrendipine as the internal standard (IS). After extraction of the plasma samples by ether-n-hexane (3:1, v/v), NIF, DNIF and the IS were subjected to LC/MS/MS analysis using electro-spray ionization (ESI). Chromatographic separation was performed on a Hypersil BDS C(18) column (50 mm x 2.1 mm, i.d., 3 microm). The method had a chromatographic running time of approximately 2.5 min and linear calibration curves over the concentrations of 0.5-100 ng/mL for NIF and DNIF. The recoveries of the one-step liquid extraction method were 81.3-89.1% for NIF and 71.6-80.4% for DNIF. The lower limit of quantification (LLOQ) of the analytical method was 0.5 ng/mL for both analytes. The intra- and inter-day precision was less than 15% for all quality control samples at concentrations of 2, 10, and 50 ng/mL. The validated LC/MS/MS method has been successfully used to study pharmacokinetic interactions of NIF with the herbal antidepressant St. John's wort in healthy volunteers. These results indicated that the developed LC/MS/MS method was efficient with a significantly shorter running time (2.5 min) for NIF and DNIF compared to those methods previously reported in the literature. The presented LC/MS/MS method had acceptable accuracy, precision and sensitivity and was used in a clinical pharmacokinetic interaction study of NIF with St. John's wort, a known herbal inducer of CYP3A4. St. John's wort was shown to induce NIF metabolism with increased plasma concentrations of DNIF.     

HPLC-MS/MS法测定人血浆中草乌甲素的浓度及生物等效性研究(英文)

本文建立了一种快速、高灵敏的HPLC-MS/MS法用于检测人血浆中的草乌甲素浓度。血浆样品采用沃特斯HLB小柱进行固相萃取,汉邦C18色谱柱(150 mm×4.6 mm,5μm)进行分离,流动相为甲醇∶水(85∶15,v/v),水相含10 mmol/L的醋酸铵和0.1%的甲酸。采用ESI源和多反应监测(MRM)的方式进行检测,草乌甲素及内标的反应离子对分别为644.4/584.4和237.2/194.2,草乌甲素血药浓度在0.010～1.0 ng/mL范围内线性关系良好,最低定量限为0.010 ng/mL可以满足口服0.4 mg草乌甲素后血药浓度的检测,日内日间及质控样品精密度及准确度均在允许范围内。本检测方法被成功的应用在中国健康志愿者生物等效性研究中,20名志愿者口服0.4 mg草乌甲素试验制剂和参比制剂后主要药代动力学参数分别如下:Cmax(0.325±0.110),(0.323±0.115)ng/mL;AUC0-16(1.627±0.489),(1.732±0.556)ng.h/mL;AUC0-∞(1.730±0.498),(1.831±0.562)ng.h/mL;t1/2(4.26±0.95),(3.80±0.90)h;Tmax(1.34±0.54),(1.83±0.99)h。     

An HPLC method for the pharmacokinetic study of daidzein-loaded nanoparticle formulations after injection to rats

This study was aimed at developing a simple HPLC method for the detection of daidzein in rat plasma. Daidzein was extracted from rat plasma with ethylparaben as internal standards (IS). Chromatographic separation of daidzein and IS was achieved by a Dikma Dimonsil C18 column (200 mm × 4.6mm) with the mobile phase consisting of methanol-water (55:45, v/v) at a flow rate of 1.0 mL/min. The injection volume was 20 μL and the detecting wavelength was 249 nm. The calibration curve was linear over a concentration range from 0.05 to 5 μg/mL, and the accuracy was within a range of 93.4-126.2%. This HPLC method was applied successfully to the pharmacokinetic study of two kinds of daidzein-loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (D-NPs) and daidzein suspension after intravenous injection in rats. Significant differences in main pharmacokinetic parameters of daidzein suspension and D-NPs were observed.     

Development and validation of a highly sensitive and robust LC-MS/MS with electrospray ionization method for simultaneous quantitation of itraconazole and hydroxyitraconazole in human plasma: application to a bioequivalence study

A highly sensitive and specific LC-MS/MS method has been developed for simultaneous estimation of itraconazole (ITZ) and hydroxyitraconazole (OH-ITZ) with 500 microL of human plasma using fluconazole as an internal standard (IS). The API-4000 LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Solid phase extraction process was used to extract ITZ, OH-ITZ and IS from human plasma. The total run time was 3.0 min and the elution of ITZ, OH-ITZ and IS occurred at 2.08 min, 1.85 min and 1.29 min, respectively; this was achieved with a mobile phase consisting of 0.2% (v/v) ammonia solution:acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a HyPurity C(18) (50 mm x 4.6 mm, 5 microm) column. The developed method was validated in human plasma with a lower limit of quantitation of 0.50 ng/mL for both ITZ and OH-ITZ. A linear response function was established for the range of concentrations 0.5-263 ng/mL (r>0.998) for both ITZ and OH-ITZ. The intra- and inter-day precision values for ITZ and OH-ITZ met the acceptance as per FDA guidelines. ITZ and OH-ITZ were stable in the battery of stability studies, viz., bench-top, auto-sampler, dry extract and freeze/thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans.     

Rapid determination of gefitinib and its main metabolite, O-desmethyl gefitinib in human plasma using liquid chromatography-tandem mass spectrometry

A novel, rapid and specific liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous quantification of gefitinib and its predominant metabolite, O-desmethyl gefitinib in human plasma. Chromatographic separation of analytes was achieved on an Alltima C18 analytical HPLC column (150 mm × 2.1 mm, 5 μm) using an isocratic elution mode with a mobile phase comprised acetonitrile and 0.1% formic acid in water (30:70, v/v). The flow rate was 300 μL/min. The chromatographic run time was 3 min. The column effluents were detected by API 4000 triple quadrupole mass spectrometer using electrospray ionization (ESI) in positive mode. Linearity was demonstrated in the range of 5-1000 ng/mL for gefitinib and 5-500 ng/mL for O-desmethyl gefitinib. The intra- and inter-day precisions for gefitinib and O-desmethyl gefitinib were ≤10.8% and the accuracies ranged from 89.7 to 104.7% for gefitinib and 100.4 to 106.0% for O-desmethyl gefitinib. This method was used as a bioanalytical tool in a phase I clinical trial to investigate the possible effect of hydroxychloroquine on the pharmacokinetics of gefitinib. The results of this study enabled clinicians to ascertain the safety of the combination therapy of hydroxychloroquine and gefitinib in patients with advanced (Stage IIIB-IV) non-small cell lung cancer (NSCLC).     

Analysis of tamoxifen and its metabolites in human plasma by gas chromatography-mass spectrometry (GC-MS) using selected ion monitoring (SIM)

A method for the analysis of tamoxifen and its metabolites in plasma from tamoxifen treated breast cancer patients, by capillary GC-MS using selected ion monitoring has been developed. Metabolite extraction was carried out on a Sep-pak C18 cartridge and metabolite purification by selective ion exchange chromatographic steps. Satisfactory recovery of radioactive standards through the extraction and purification steps was obtained. The method was shown to be accurate and precise with precision coefficient of variation values ranging from 4.3-11% for tamoxifen and its metabolites. Tamoxifen, 4-hydroxytamoxifen, metabolite Y and N-desmethyltamoxifen were identified with certainty in patient plasma on the basis of GC relative retention times and mass spectral comparison with authentic standards; because of their low abundance in plasma cis-metabolite E and 3,4-dihydroxytamoxifen could only be tentatively identified but identical GC behaviour and a satisfactory comparison of the abundance of key fragment ions was achieved. The tamoxifen and metabolite concentration ranges (ng X ml-1) in the group of patients who received 40 or 80 ng tamoxifen for 14 days were tamoxifen, 307-745; N-desmethyltamoxifen, 185-491; 4-hydroxytamoxifen, 1.4-2.5; 3,4-dihydroxytamoxifen, 0.7-2.0; metabolite Y, 19.0-112; and metabolite E1, 0.9-2.0.     

Effects of tamoxifen and 4-hydroxytamoxifen on the pNR-1 and pNR-2 estrogen-regulated RNAs in human breast cancer cells

The estrogenic and antiestrogenic activities of tamoxifen and 4-hydroxytamoxifen have been measured on the expression of two estrogen-regulated RNAs (pNR-1 and pNR-2) in the MCF7 human breast cancer cell line cultured in phenol red-free medium. The two antiestrogens increased the level of the pNR-1 RNA to about 80% of the estradiol-induced level, and the induction by estradiol was not significantly antagonized by either antiestrogen. In contrast, the pNR-2 mRNA was only increased to about 10% of the estradiol-induced level, and its induction by estradiol was antagonized by both tamoxifen and 4-hydroxytamoxifen. Thus, the two RNAs respond in dramatically different ways to these antiestrogens. 4-Hydroxytamoxifen and estradiol have similar affinities for the estrogen receptor; however, the induction of both RNAs by 4-hydroxytamoxifen required a 10-fold higher concentration than estradiol for maximum agonist activity, and a 500-fold molar excess was required to antagonize the induction by estradiol. Tamoxifen has a 20-100-fold lower affinity than estradiol for the estrogen receptor. A 200-fold higher concentration was required for maximum agonist activity and a 10,000-fold molar excess to antagonize the induction by estradiol. These results emphasize the complexity of antiestrogen action in human breast cancer cells.     

Development and validation of a LC-ESI-MS/MS method for the determination of swertiamarin in rat plasma and its application in pharmacokinetics

A new LC-ESI-MS/MS assay method has been developed and validated for the quantification of swertiamarin, a representative bioactive substance of Swertia plants, in rat plasma using gentiopicroside, an analog of swertiamarin on chemical structure and chromatographic action, as the internal standard (IS). The swertiamarin and IS were extracted from rat plasma using solid-phase extraction (SPE) as the sample clean-up procedure, and they were chromatographed on a narrow internal diameter column (Agilent ZORBAX ECLIPSE XDB-C(18) 100 mm × 2.1 mm, 1.8 μm) with the mobile phase consisting of methanol and water containing 0.1% acetic acid (25:75, v/v) at a flow rate of 0.2 mL/min. The detection was performed on an Agilent G6410B tandem mass spectrometer by negative ion electrospray ionisation in multiple-reaction monitoring mode while monitoring the transitions of m/z 433 [M+CH(3)COO](-)→179 and m/z 415 [M+CH(3)COO](-)→179 for swertiamarin and IS, respectively. The lower limit of quantification (LLOQ) was 5 ng/mL within a linear range of 5-1000 ng/mL (n=7, r(2)≥0.994), and the limit of detection (LOD) was demonstrated as 1.25 ng/mL (S/N≥3). The method also afforded satisfactory results in terms of sensitivity, specificity, precision (intra- and inter-day), accuracy, recovery, freeze/thaw, long-time stability and dilution integrity. This method was successfully applied to determination of the pharmacokinetic properties of swertiamarin in rats after oral administration at a dose of 20 mg/kg. The following pharmacokinetic parameters were obtained (mean): maximum plasma concentration, 1920.1 ng/mL; time to reach maximum plasma concentration, 0.945 h; elimination half-time, 1.10h; apparent total clearance, 5.638 L/h/kg; and apparent volume of distribution, 9.637 L/kg.     

Direct determination of tamoxifen and its four major metabolites in plasma using coupled column high-performance liquid chromatography

A rapid, rugged and fully automated method has been developed for the determination of tamoxifen and its major metabolites in plasma. The system is based upon an in-line extraction process combined with column switching to a coupled analytical column. The plasma sample is deproteinated by the addition of acetonitrile before injection onto a semi-permeable surface (SPS) cyano guard column (1.0 × 0.46 cm I.D.). After washing the guard column briefly with water, the sample is eluted with a mobile phase composed of 35% acetonitrile in 20 mM potassium phosphate buffer (pH 3). The eluent is directed through a cyano analytical column (25 × 0.46 cm I.D.) and a photochemical reactor where the analytes are converted to highly fluorescent phenanthrene derivatives. Tamoxifen, 4-hydroxytamoxifen, N-desdimethyltamoxifen, N-desmethyltamoxifen and tamoxifen-ol are eluted in that order at a flow-rate of 1.0 ml/min. The method has been validated for use in a clinical study utilizing tamoxifen in the treatment of recurrent cerebral astrocytomas.