Quantitation of ursolic acid in human plasma by ultra performance liquid chromatography tandem mass spectrometry and its pharmacokinetic study

Ursolic acid is a hydroxy pentacyclic triterpene, which proved to have sedation, anti-inflammatory, antibacterial, antiulcer and anti-cancer activities. An ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method with high selectivity, sensitivity and throughput has been established and validated for quantitation of total ursolic acid in human plasma. Plasma samples were pretreated by liquid-liquid extraction with ethyl acetate and were chromatographed by an ACQUITY UPLC BEH C(8) column (100 mm×2.1 mm, I.D., 1.7 μm) using mobile phase consisting of acetonitrile and 10 mM ammonium formate (90:10, v/v) at 0.2 mL/min. The duration of chromatography analysis was 3 min. The multiple reaction monitoring (MRM) was performed at m/z 455.1→455.0 for ursolic acid and m/z 469.3→425.2 for glycyrrhetinic acid (internal standard, IS) in the negative ion mode with electrospray ionization (ESI) source. The assay showed good linearity over the range of 10-5000 ng/mL for ursolic acid in human plasma with a lower limit of quantitation of 10 ng/mL. The mean extraction recovery was 73.2±4.5% and the matrix ion suppression ranged from -11.4% to -5.6%. The intra- and inter-day precisions were less than 7.0% and 7.2%, respectively, and the accuracy was within ±2.0%. Ursolic acid was stable during the analysis and the storage period. The validated method has been successfully applied to a pharmacokinetic study after intravenous infusion of Ursolic Acid Nano-liposomes to healthy volunteers.     

Simultaneous UFLC-ESI-MS/MS determination of piperine and piperlonguminine in rat plasma after oral administration of alkaloids from Piper longum L.: application to pharmacokinetic studies in rats

The alkaloids from Piper longum L. showed protective effects on Parkinson's disease models in our previous study and piperine and piperlonguminine were the two main constituents in the alkaloids. The present study aimed at developing a rapid, sensitive, and accurate UFLC-ESI-MS/MS method and validating it for the simultaneous determination of piperine and piperlonguminine in rat plasma using terfenadine as the internal standard. The analytes and internal standard (IS) were extracted from rat plasma using a simple protein precipitation by adding methanol/acetonitrile (1:1, v/v). A Phenomenex Gemini 3 u C18 column (20 mm × 2.00 mm, 3 μm) was used to separate the analytes and IS using a gradient mode system with a mobile phase consisting of water with 0.1% formic acid (mobile phase A) and acetonitrile with 0.1% formic acid (mobile phase B) at a flow rate of 0.4 mL/min and an operating column temperature of 25°C. The total analytical run time was 4 min. The detection was performed using the positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode with transitions at m/z 286.1-201.1 for piperine, m/z 274.0-201.1 for piperlonguminine, and m/z 472.4-436.4 for the IS. The calibration curves were both linear (r>0.995) over a concentration range of 1.0 to 1000 ng/mL; the lower limit of quantification (LLOQ) was 1.0 ng/mL for both piperine and piperlonguminine. The intra-day and inter-day precisions (RSD %) were <12.1%, accuracies ranged from 86.6 to 120%, and recoveries ranged from 90.4 to 108%. The analytes were proven stable in the short-term, long-term, and after three freeze-thaw cycles. The method was successfully applied to pharmacokinetic studies of piperine and piperlonguminine in rats after oral administration of alkaloids from P. longum L.     

Application of a liquid chromatography/tandem mass spectrometry method to pharmacokinetic study of mangiferin in rats

A simple, rapid and accurate liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed and validated for quantification of mangiferin in rat plasma. After the addition of the internal standard (IS) paracetamol, plasma samples were pretreated by protein precipitation. Chromatographic separation was carried out on a C(18) column by isocratic elution with methanol-acetonitrile-1% acetic acid (40:3:57, v/v/v). The detection was performed on a Sciex API 3000 LC/MS/MS with TurboIonSpray ionization (ESI) inlet in the positive ion MRM mode. Good linearity was achieved over the concentration range of 3.01-601 ng/mL. Intra- and inter-day precisions were less than 9.1%, and accuracy ranged from 100.5% to 104.0%. The pharmacokinetic profiles of free mangiferin at three dose levels and mangiferin in Zhimu decoction and Zhimu-Huangbai decoction were studied for the first time in rats by this method. After single intragastric administration of free mangiferin 17.5, 35 and 70 mg/kg, C(max) and AUC increased but non-proportional to the doses. At the same dose level (35 mg/kg), C(max) and AUC of mangiferin in two decoctions were significantly higher than the corresponding values of free mangiferin.     

Determination of 5-aminoimidazole-4-carboxamide in human plasma by ion-pair extraction and LC-MS/MS

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was established for the determination of 5-aminoimidazole-4-carboxamide (AICA) in human plasma. The method included a solvent extraction of AICA as an ion pair with 1-pentanesulfonate ion and a separation on a Hypersil ODS2 column with the mobile phase of methanol-water (68:32, v/v). Determination was performed using an electrospray ionization source in positive ion mode (ESI(+)). Multiple reaction monitoring (MRM) was utilized for the detection monitoring m/z at 127-->110 for AICA, and 172-->128 for IS. The calibration curve was linear within a range from 20 to 2000 ng/mL and the limit of quantity for AICA in plasma was 20 ng/mL. RSD of intra-assay and inter-assay were no more than 5.90% and 5.65%.     

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.     

A rapid, sensitive and selective liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry method for determination of fenretinide (4-HPR) in plasma

A simple and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method using an atmospheric pressure chemical ionization source (APCI) for the quantification of fenretinide (4-HPR) in mouse plasma was developed and validated. After a simple protein precipitation of plasma sample by acetonitrile, 4-HPR was analyzed by LC-APCI-MS/MS. High-performance liquid chromatography (HPLC) separation was conducted on a Hypurity C18 column (50mmx2.1mm, 5microm) with a flow rate 0.60mL/min using a gradient mobile phase comprised of 0.05% formic acid in water (A) and methanol (B), and a run time of 4.5min. The elimination of a tedious sample preparation process and a shorter run time substantially reduced total analysis time. The method was linear over the range 0.5-100ng/mL, with r>0.998. The intra- and inter-assay precisions were 1.4-9.2% and 5.1-8.2%, respectively, and the intra- and inter-assay accuracies were 93.9-98.6% and 92.7-95.3%, respectively. The absolute recoveries were 90.3% (1.5ng/mL), 97.0% (7.5ng/mL) and 92.1% (75.0ng/mL) for 4-HPR, and 99.1% for the internal standard (150ng/mL). The analytical method had excellent sensitivity using a small sample volume (30microL) with the lower limit of quantification (LLOQ) 0.5ng/mL. This method is robust and has been successfully employed in a pharmacokinetic study of 4-HPR in a mouse xenograft model of neuroblastoma.     

Rapid and Simultaneous Quantification of Levetiracetam and Its Carboxylic Metabolite in Human Plasma by Liquid Chromatography Tandem Mass Spectrometry

A simple liquid chromatography tandem mass spectrometry method was developed and validated according to the guidelines of the US Food and Drug Administration and the European Medicines Agency for a simultaneous quantification of levetiracetam (LEV) and its metabolite, UCB L057 in the plasma of patients. A 0.050 mL plasma sample was prepared by a simple and direct protein precipitation with 0.450 mL acetonitrile (ACN) containing 1 µg/mL of internal standard (IS, diphenhydramine), then vortex mixed and centrifuged. A 0.100 mL of the clear supernatant was diluted with 0.400 mL water and well mixed. A 0.010 mL of the resultant solution was injected into an Agilent Zorbax SB-C18 (2.1 mm×100 mm, 3.5 µm) column with an isocratic elution at 0.5 mL/min using a mixture of 0.1% formic acid in water and ACN (40∶60 v/v). Detection was performed using an AB Sciex API 3000 triple quadrupole mass spectrometer, equipped with a Turbo Ion Spray source, operating in a positive mode: LEV at transition 171.1>154.1, UCB L057 at 172.5>126.1, and IS at 256.3>167.3; with an assay run time of 2 minutes. The lower limit of quantification (LLOQ) for both LEV and UCB L057 was validated at 0.5 µg/mL, while their lower limit of detection (LOD) was 0.25 µg/mL. The calibration curves were linear between 0.5 and 100 µg/mL for both analytes. The inaccuracy and imprecision of both intra-assay and inter-assay were less than 10%. Matrix effects were consistent between sources of plasma and the recoveries of all compounds were between 100% and 110%. Stability was established under various storage and processing conditions. The carryovers from both LEV and UCB L057 were less than 6% of the LLOQ and 0.13% of the IS. This assay method has been successfully applied to a population pharmacokinetic study of LEV in patients with epilepsy.     

Simultaneous quantification of 17α-OH progesterone, 11-deoxycortisol, Δ4-androstenedione, cortisol and cortisone in newborn blood spots using liquid chromatography-tandem mass spectrometry

Adrenal steroid profiling, including 17α-OH progesterone (17OHP), 11-deoxycortisol (S), Δ4-androstenedione (Δ4-A) and cortisol (F) in blood spots by tandem mass spectrometry, is used for newborn screening to detect congenital adrenal hyperplasia (CAH). Pre-analytical sample processing is critical for assay specificity and accuracy; however, it is laborious and time-consuming. This study describes the development and validation of a new Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) method for the simultaneous quantification of five steroids: 17OHP, S, Δ4-A, F and cortisone (E) in blood spots from newborns. Whole blood was eluted from a 5.00 mm dried blood spot by an aqueous solution containing the deuterium-labeled internal standards d8-17OHP and d4-cortisol. The steroids extracted from blood spot into aqueous solution were subsequently purified via Extelut mini NT1 column using diethylether. The extracts were evaporated and quantified using LC-MS/MS. The detection limit was 0.25 ng/mL for 17OHP and S, 0.4 ng/mL for Δ4-A and 0.5 ng/mL for F and E. The limit of quantification was 0.5 ng/mL for 17OHP, S and Δ4-A and 1 ng/mL for F and E. Precision for 17OHP, S, Δ4-A at concentrations of 0.5, 2, and 8 ng/mL (n=5) in fortified steroid free serum samples was 1.3-3.5% (intra-assay CV) and 7-14.8% (inter-assay CV). Precision for F and E at concentrations of 5 and 20 ng/mL was 1.5-4.8% (intra-assay, CV%) and 6-15% (inter-assay, CV%). Accuracy was calculated at concentrations of 0.5, 2, and 8 ng/mL for 17OHP, S and Δ4-A and ranged from -0.3 to 0.2%, while for F and E it ranged from -3.2 to 0.2%. Relative recoveries at concentration 2 ng/mL and 8 ng/mL for 17OHP, S, Δ4-A and at 5 ng/mL and 20 ng/mL for F and E ranged from 55% to 80%. Reference intervals were estimated for all steroids in newborns (on day 3). The steroid profile assay herein described is sensitive, specific and accurate and involves a simple pre-analytical sample manipulation; it is therefore suitable for routine analysis and provides data for samples within normal range as well as those with elevated levels. For the first time to our knowledge, cortisone levels are reported in dried blood spots from newborns.     

Sensitive high-performance liquid chromatographic method using coulometric electrode array detection for measurement of phytoestrogens in dried blood spots

As the epidemiological and physiological investigation of isoflavones and lignans expands, the need for sensitive methods for analyzing large numbers of samples intensifies. We have developed a method using high-performance liquid chromatography (HPLC) equipped with a coulometric electrode array detector for separation and sensitive detection of daidzein (Da), equol (Eq), genistein (Ge) and enterolactone (Enl) in dried blood spots (DBS). Detection limits ranged from 4.5 pg or 0.09 ng/mL (Eq) to 19 pg or 0.38 ng/mL (Ge) on column. Signal linearities ranged from detection limits to 200 ng/mL (Eq, Enl) and 600 ng/mL (Da, Ge) sample concentration. Correlations between DBS and serum concentrations were 0.66 (Enl), 0.88 (Eq), 0.98 (Ge) and 0.99 (Da). Intra-assay coefficients of variation (CVs) were less than 8% and inter-assay CVs ranged from 2.4 to 20.2% for Da, Eq and Ge for three levels of controls. Enl intra-assay CV was 13.6% for the low pooled control. Analytic recovery ranged from 87% (inter-assay Ge) to 98% (inter-assay Enl). DBS concentrations of Da, Ge and Eq were stable for at least 8 weeks at 4 and 25 degrees C, and at 37 degrees C for at least 5 weeks, with Enl showing greater variability at all temperatures but relative stability for 7 weeks. Measurement of samples from 135 perimenopausal Japanese women consuming habitual diets in Kyoto and Fukushima prefectures showed the former to have the expected lower concentrations of Da and Eq (416 and 87 nM) as well as Enl (49 nM) compared to the latter locale (566, 145 and 72 nM, respectively). This method could be useful in large epidemiological research or detailed physiological studies.     

Development and validation of a quantitative assay for the measurement of miltefosine in human plasma by liquid chromatography-tandem mass spectrometry

A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the quantification of miltefosine is presented. A 250 microL human EDTA plasma aliquot was spiked with miltefosine and extracted by a solid-phase extraction method. Separation was performed on a Gemini C18 column (150 mm x 2.0 mm I.D., 5 microm) using an alkaline eluent. Detection was performed by positive ion electrospray ionization followed by triple-quadrupole mass spectrometry. The assay has been validated for miltefosine from 4 to 2000 ng/mL using 250 microL human EDTA plasma samples. Results from the validation demonstrate that miltefosine can be accurately and precisely quantified in human plasma. At the lowest level, the intra-assay precision was lower than 10.7%, the inter-assay precision was 10.6% and accuracies were between 95.1 and 109%. This assay is successfully used in a clinical pharmacokinetic study with miltefosine.     

Simultaneous determination of four active alkaloids from a traditional Chinese medicine Corydalis saxicola Bunting. (Yanhuanglian) in plasma and urine samples by LC-MS-MS

A sensitive rapid method for the simultaneous determination of four major active alkaloids (dehydrocavidine, coptisine, dehydroapocavidine, and tetradehydroscoulerine, in abbreviation thereafter called YHL-I, YHL-II, YHL-III, and YHL-IV, respectively) from a Chinese traditional medicine Corydalis saxicola Bunting. (Yanhuanglian) in rat plasma and urine was established and validated. The assay for these substances in plasma and urine was based on HPLC coupled with tandem mass spectrometry (MS/MS) detection using multiple reaction monitoring mode (MRM) with berberine and clenbuterol as internal standards. The plasma and urine sample were deproteinated by adding methanol prior to liquid chromatography where separation was performed on a Luna column (5 microm, 100 x 2.00 mm) and an Agilent Zorbax SB-C18 guard column (5 microm, 20 x 4 mm). The method was validated with the concentration range 1-1000 ng/mL in plasma and 10-1000 ng/mL in urine for the four test compounds, and the calibration curves were linear with correlation coefficients >0.999. The lowest limits of quantitation for all four substances were 1 ng/mL in 0.1 mL rat plasma and 10 ng/mL in 0.1 mL urine. The intra-assay accuracy and precision in plasma ranged from 88.1 to 115.7% and 1.4 to 10.8%, respectively, while inter-assay accuracy and precision for YHL-I, YHL-II, YHL-III, and YHL-IV ranged from 96.2 to 113.2% and 0.4 to 16.9%, respectively. The intra-assay accuracy and precision for YHL-I, YHL-II, YHL-III, and YHL-IV in rat urine ranged from 96.1 to 112.9% and 1.2 to 8.3%, respectively, while inter-assay accuracy and precision ranged from 95.0 to 106.8% and 2.2 to 10.3%, respectively. The method was further applied to assess pharmacokinetics and urine excretion of the four alkaloids after oral and intravenous administration to rats. Practical utility of this new LC-MS-MS method was confirmed in pilot pharmacokinetic studies in rats following both intravenous and oral administration.     

Human plasma quantification of droperidol and ondansetron used in preventing postoperative nausea and vomiting with a LC/ESI/MS/MS method

An analytical method based upon liquid chromatography coupled to ion trap mass spectrometry (MS) detection with electrospray ionization interface has been developed for the simultaneous identification and quantification of droperidol and ondansetron in human plasma. The two drugs were isolated from 0.5 mL of plasma using a basic liquid-liquid extraction with diethyl ether/heptane (90/10, v/v) and tropisetron and haloperidol as internal standards, with satisfactory extraction recoveries. They were separated on a 5-μm C(18) Highpurity column (150 mm×2.1 mm I.D.) maintained at 30°C. The elution was achieved isocratically with a mobile phase of 2 mM HCOONH(4) pH 3.8 buffer/acetonitrile (60/40, v/v) at a flow rate of 200 μL/min. Data were collected either in full-scan MS mode at m/z 100-450 or in full-scan MS-MS mode, selecting the [M+H] (+) ion at m/z=294.0 for ondansetron, m/z=285.2 for tropisetron, m/z=380.0 for droperidol and m/z=376.0 for haloperidol. The most intense daughter ion of ondansetron (m/z=212.0) and droperidol (m/z=194.0) were used for quantification. Retention times for tropisetron, ondansetron, droperidol and haloperidol were 2.50, 2.61, 3.10 and 4.68 min, respectively. Calibration curves were linear for both compounds in the 0.50-500 ng/mL range. The limits of detection and quantification were 0.10 ng/mL and 0.50 ng/mL, respectively. The intra- and inter-assay precisions were lower than 6.4% and intra- and inter-assay recoveries were in the 97.6-101.9% range for the three 3, 30 and 300 ng/mL concentrations. This method allows simultaneous and rapid measurement of droperidol and ondansetron, which are frequently co-administrated for the prevention of postoperative nausea and vomiting.     

Quantitative determination of olanzapine in rat brain tissue by high-performance liquid chromatography with electrochemical detection

A sensitive assay was developed for the measurement of olanzapine in rat brain tissue using HPLC with electrochemical detection. The assay has a lower limit of quantitation of 0.5 ng/ml in tissue homogenate and utilizes a liquid–liquid extraction followed by reversed-phase HPLC for the quantitative analysis of olanzapine. The method provided a linear response for olanzapine over a concentration range of 0.5–100 ng/ml with a coefficient of determination (r²) greater than 0.9995. The extraction efficiencies of olanzapine and internal standard (LY170158) were greater than 82% in brain tissue. The intra-assay and inter-assay relative errors ranged from −5.38 to 17.60% and −3.25 to 10.53%, respectively. The intra-assay and inter-assay RSD values were in the range of 1.12 to 6.96% and 3.78 to 6.68%. Long-term stability studies showed that brain tissue homogenate samples spiked with olanzapine and internal standard are stable at −70°C for at least 110 days. However, a room temperature stability study showed that olanazapine was not stable in brain homogenate if the sample was exposed at 25°C longer than 2 h. This method has been used for the study of the disposition and pharmacokinetics of olanzapine in male Sprague–Dawley rats.     

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。     

Rapid quantification of the delta-opioid receptor selective enkephalin DPDPE in canine cerebrospinal fluid by liquid chromatography--mass spectrometry

An atmospheric pressure ionization liquid chromatographic-mass spectrometric assay was developed and validated for the determination of D-penicillamine(2,5) enkephalin (DPDPE) in cerebrospinal fluid (CSF) from dog. DPDPE and internal standard (D-Ala(2),D-Leu(5) enkephalin=DADLE) were isolated from CSF by reversed-phase C(18) solid-phase extraction with ZipTip micro-cartridges. Aliquots of extracted eluate were injected onto an Agilent Zorbax SB C(18) column (30 x 2.2 mm; 3.5 microm) at a flow-rate of 0.4 ml/min. The isocratic mobile phase of methanol-10 mM ammonium formate (pH 3) (75:25, v/v) was then diverted to waste for 45 s after injection, after which time flow was directed to the single quadrupole mass spectrometer. DPDPE was detected by positive mode selected ion monitoring. Standard curves were linear (r(2)> or =0.991) over the concentration range 1-1000 ng/ml. The efficiency of extraction recovery was greater than 97%, and the intra-assay and inter-assay precisions were within 9% relative standard deviation. DPDPE and the internal standard were stable in the injection solvent at 4 degrees C for at least 48 h. The assay was applied to the pharmacokinetic study of intrathecal DPDPE administration in the dog animal model.     

Development and validation of a high-performance liquid chromatography-tandem mass spectrometry for the determination of penciclovir in human plasma: application to a bioequivalence study

We developed and validated a simple high-performance liquid chromatography (HPLC) coupled with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection system for determining penciclovir (active metabolite of famciclovir) levels in human plasma using acyclovir as an internal standard (IS). Acquisition was performed in multiple reaction monitoring (MRM) mode by monitoring the transitions: m/z 254.00>152.09 for penciclovir and m/z 226.00>152.09 for IS. The analytes were chromatographed on a Capcellpak MGII C(18) reversed-phase chromatographic column by isocratic elution using 30% methanol and 70% Milli-Q water containing 10 mM ammonium formate (adjusted to pH 3.1 with formic acid). Results were linear over the studied range (0.05-10 microg/ml) with r(2)=0.9999, and the total analysis time for each run was 2 min. Intra- and inter-assay precisions were 2.3-7.8 and 3.7-7.5%, respectively, and intra- and inter-assay relative errors (RE) were 2.0-8.4 and 1.9-9.1%, respectively. The lower limit of quantification (LLOQ) was 0.05 microg/ml. At this concentration mean intra- and inter-assay precisions were 7.8 and 7.5%, respectively, and mean intra- and inter-assay relative errors were 2.2 and 9.1%, respectively. Penciclovir was found to be stable in plasma samples under short-, long-term storage and processing conditions. The developed assay was successfully applied to a bioequivalence study of penciclovir administered as a single oral dose (500 mg as famciclovir) to healthy volunteers.     

High performance liquid chromatography-electrospray ionization mass spectrometric determination of isosorbide 5-mononitrate in human plasma

A novel, selective and sensitive high performance liquid chromatography-mass spectrometric (HPLC-MS) method has been developed for the determination of isosorbide 5-mononitrate (5-ISMN) in human plasma. With acetaminophen as internal standard, sample pretreatment involved one-step extraction with diethyl ether of 0.5 mL plasma. Analysis was performed on an ACQUITY UPLC BEH C(18) column (100 mm x 2.1mm, 1.7 microm) with mobile phase consisting of acetonitrile-water (20:80, v/v). The detection was carried out by means of electrospray ionization mass spectrometry in negative ion mode with selected ion recording (SIR). Standard curves were linear (r(2)> or =0.99) over the concentration range of 1.04-1040 ng/mL. The lower limit of quantification (LLOQ) was 1.04 ng/mL. The intra- and inter-day precisions (RSDs) were less than 8.6% and 13.4%, respectively, and the accuracy (RE) was within +/-0.45%. The method herein described was fully validated and successfully applied to the pharmacokinetic study of 5-ISMN in compound extended-release tablets in 18 healthy male volunteers after oral administration.     

Quantitative determination of forty-eight antidepressants and antipsychotics in human serum by HPLC tandem mass spectrometry: a multi-level, single-sample approach

This method describes the simultaneous determination of amisulpride, amitriptyline, aripiprazole, benperidol, chlorpromazine, chlorprothixene, citalopram, clomipramine, clozapine, desipramine, doxepin, fluoxetine, flupentixol, fluphenazine, fluvoxamine, haloperidol, hydroxyrisperidone, imipramine, levomepromazine, maprotiline, mianserine, mirtazapine, moclobemide, norclomipramine, nordoxepin, norfluoxetine, nortriptyline, O-desmethylvenlafaxine, olanzapine, opipramol, paroxetine, perazine, perphenazine, pimozide, pipamperone, quetiapine, reboxetine, risperidone, sertraline, sulpiride, thioridazine, trazodone, trimipramine, venlafaxine, viloxazine, ziprasidone, zotepine and zuclopenthixol with a single sample/triple injection approach. Drugs were assigned to subgroups covering low, medium and high concentrations (overall range of therapeutic levels to be considered: 0.5-2000 ng/mL) by further dilution of the supernatant obtained after the first protein precipitation. Chromatographic separation was necessary for isobaric mass fragments and performed on a monolithic C18 column (50mmx4.6mm) with methanol gradient and 5mM acetate buffer at pH 3.9. The injection interval was 8 min. A set of three internal standards was used for quantification of drugs with widely varying hydrophobicity. After electrospray ionization positive ion fragments were detected in the multiple reaction monitoring (MRM) mode with an API 4000 tandem mass spectrometer. Regression parameters of calibration curves and limits of quantification showed good covering of therapeutic and subtherapeutic ranges with an average correlation coefficient of 0.9988. Imprecision and inaccuracy measures were prepared for intra- and inter-assay comparisons at three concentration ranges in all subgroups. Average coefficients of variation were 6.1% for intra-assay and 7.4% for inter-assay comparisons, while average deviations from spiked concentrations were 4.8% for intra-assay and 4.2% for inter-assay comparisons, respectively. Recovery rates, measured as the percent recoveries of spiked serum samples against standard solutions without serum matrix, varied between 92 and 111%, with an average of 101%. As the only exception, the olanzapine response was much higher (185%) in serum matrix than in matrix-free controls.     

Direct-injection HPLC method of measuring micafungin in human plasma using a novel hydrophobic/hydrophilic hybrid ODS column

A direct-injection HPLC-based method has been developed for determining amounts of micafungin in human plasma using a novel hydrophobic/hydrophilic hybrid ODS column. The method is easy to perform and requires only 10 μL of a filtered plasma sample. The chromatographic separations were carried out with a gradient mode. The fluorescence detection wavelengths of excitation and emission were set at 273 nm and 464 nm, respectively. Retention times for micafungin and IS were 22.4 and 23.7 min, respectively. Micafungin and FR195743 (IS) peaks were completely separated with little tailing, and no interference was observed. The calibration curve of micafungin showed good linearity in the range of 0.5-20.0 μg/mL (r(2)=1.00). The intra-day accuracy ranged from -4.5 to 5.3%. The inter-day accuracy ranged from -9.8 to 1.5%. The precisions were less than 10%. This method is useful for the determination of micafungin in human plasma.     

Simultaneous determination of etoposide and a piperine analogue (PA-1) by UPLC–qTOF-MS: Evidence that PA-1 enhances the oral bioavailability of etoposide in mice

In the present investigation, a UPLC–qTOF-MS/MS method has been developed for the simultaneous determination of etoposide and a piperine analogue, namely, 4-ethyl 5-(3,4-methylenedioxyphenyl)-2E,4E-pentadienoic acid piperidide (PA-1). The analytes were separated on a reverse phase C18 column using methanol–water (72:28, v/v) mobile phase with a flow rate of 250 μL/min. The qTOF-MS was operated under multiple reaction monitoring mode using electro-spray ionization (ESI) technique with positive ion polarity. The major product ions for etoposide and PA-1 were at m/z 185.1350 and 164.1581, respectively. The recovery of the analytes from mouse plasma was optimized using solid phase extraction technique. The total run time was 6 min and the elution of etoposide and PA-1 occurred at 1.24 and 2.84 min, respectively. The calibration curves of etoposide as well as PA-1 were linear over the concentration range of 2–1000 ng/mL (r², 0.9829), and 1–1000 ng/mL (r², 0.9989), respectively. For etoposide intra-assay and inter-assay accuracy in terms of % bias was in between ?7.65 to +6.26, and ?7.83 to +5.99, respectively. For PA-1 intra-assay and inter-assay accuracy in terms of % bias was in between ?7.01 to +9.10, and ?7.36 to +6.71, respectively. The lower limit of quantitation for etoposide and PA-1 were 2.0 and 1.0 ng/mL, respectively. Analytes were stable under various conditions (in autosampler, during freeze–thaw, at room temperature, and under deep-freeze conditions). The method was used for a pharmacokinetic study which showed that PA-1 enhanced the oral bioavailability of etoposide in mice by 2.32-fold.