Simple High-Performance Liquid Chromatographic Assay, with Post-Column Derivatization, for Simultaneous Determination of Mycophenolic Acid and its Glucuronide Metabolite in Human Plasma and Urine

Two simple high-performance liquid chromatographic (HPLC) methods have been established for simultaneous determination of mycophenolic acid (MPA) and its glucuronide metabolite (MPAG) in human urine, and of their total and unbound forms in human plasma. For total MPA and MPAG analysis sample preparation entailed precipitation of protein with acetonitrile and isolation of the free analytes from the plasma by ultrafiltration. For urine samples, fivefold dilution with water was used. MPAG was determined by UV detection whereas MPA was quantified by fluorescence detection after post-column derivatization with 0.2 M sodium hydroxide solution. For plasma, response was found to be linearly dependent on concentration over the ranges 0.1–40 μg mL^-1 and 0.01–1 μg mL^-1 for total and free MPA, respectively, and 10–200 μg mL^-1 and 2.5–100 μg mL^-1 for total and free MPAG, respectively. For urine, linearity was observed from 0.1 to 50 μg mL^-1 for MPA and 10 to 500 μg mL^-1 MPAG in the urine before dilution. The methods reported were found to be accurate and reproducible for quantifying the level of MPA and MPAG and can thus be used for clinical pharmacokinetic studies and for therapeutic drug monitoring. Contributed equally to this work An erratum to this article is available at .     

LC–MS/MS Determination of Antihypertension Drugs in Rat Plasma and Urine: Applications to Pharmacokinetics

A sensitive, rapid and reproducible LC–MS/MS method for the determination of olmesartan (OLM), amlodipine (ALM) and hydrochlorothiazide (HCZ) in rat plasma and urine has been developed and validated. Irbesartan (IRB) was used as an internal standard. The analytes were separated on a Waters XTerra-C₁₈ column using gradient elution with acetonitrile and 10 mM ammonium acetate buffer (pH 3.5, adjusted with acetic acid) at a flow rate of 1.0 mL min^?1. The three analytes were ionized by positive ion electrospray using multiple-reaction monitoring (MRM) mode to monitor precursor?→?product ion transitions m/z 447.31?→?234.97 for OLM, 408.87?→?238.18 for AML and 290.1?→?204.85 for HCZ. The specificity, matrix effect, recovery, sensitivity, linearity, accuracy, precision, and stabilities were all validated over the concentration range 0.4–100 ng mL^?1 for AML, 0.2–100 ng mL^?1 for OLM, 0.1–100 ng mL^?1 for HCZ. The mean concentrations (C_max) are 10.32, 587, and 3.4 for OLM, ALM, and HCZ, respectively, by the oral administration of 15 mg kg^?1 of each analyte.     

Monolithic silica spin column extraction and simultaneous derivatization of amphetamines and 3,4-methylenedioxyamphetamines in human urine for gas chromatographic-mass spectrometric detection

A simple, sensitive, and specific method with gas chromatography-mass spectrometry was developed for simultaneous extraction and derivatization of amphetamines (APs) and 3,4-methylenedioxyamphetamines (MDAs) in human urine by using a monolithic silica spin column. All the procedures, such as sample loading, washing, and elution were performed by centrifugation. APs and MDAs in urine were adsorbed on the monolithic silica and derivatized with propyl chloroformate in the column. Methamphetamine-d₅ was used as an internal standard. The linear ranges were 0.01-5.0 μg mL⁻¹ for methamphetamine (MA) and 3,4-methylenedioxymethamphetamine (MDMA) and 0.02-5.0 μg mL⁻¹ for amphetamine (AP) and 3,4-methylenedioxyamphetamine (MDA) (coefficient of correlation ≧0.995). The recovery of APs and MDAs in urine was 84-94%, and the relative standard deviation of the intra- and interday reproducibility for urine samples containing 0.1, 1.0, and 4.0 μg mL⁻¹ of APs and MDAs ranged from 1.4% to 13.6%. The lowest detection limit (signal-to-noise ratio ≧ 3) in urine was 5 ng mL⁻¹ for MA and MDMA and 10 ng mL⁻¹ for AP and MDA. The proposed method can be used to perform simultaneous extraction and derivatization on spin columns that have been loaded with a small quantity of solvent by using centrifugation.     

Simultaneous Determination of Atenolol and Chlorthalidone by LC–MS–MS in Human Plasma

A simple, sensitive, selective and rapid liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous separation and quantitation of atenolol and chlorthalidone in human plasma using metoprolol and hydrochlorothiazide as internal standard. Following solid phase extraction, the analytes were separated by an isocratic mobile phase on a reversed-phase C₁₈ column and analyzed by MS in the multiple reaction-monitoring mode (atenolol in positive and chlorthalidone in the negative ion mode). The limit of quantitation for this method was 10 and 15 ng mL^?1 and the linear dynamic range was generally 10–2,050 ng mL^?1 and 15–3,035 ng mL^?1 for atenolol and chlorthalidone, respectively.     

Simultaneous LC–MS–MS Analysis of Danshensu, Salvianolic Acid B, and Hydroxysafflor Yellow A in Beagle Dog Plasma, and Application of the Method to a Pharmacokinetic Study of Danhong Lyophilized Powder for Injection

A reliable and sensitive liquid chromatographic–tandem mass spectrometric method, with rutin as internal standard, has been developed and validated for simultaneous determination of danshensu, salvianolic acid B (SAB), and hydroxysafflor yellow A (HSYA) in beagle dog plasma. Plasma samples spiked with the analytes were extracted by solid-phase extraction and the analytes were separated on a 250 × 4.6 mm i.d., 5-μm particle, C₁₈ column with methanol–acetonitrile–0.5% formic acid 20:25:55 (v/v) as mobile phase at a flow rate of 1 mL min^?1. LC–MS–MS analysis was performed with a Finnigan TSQ triple-quadrupole tandem mass spectrometer operated in negative-ion selected-reaction-monitoring mode, using electrospray ionization. The accuracy and precision of the method were acceptable and linearity was good over the range 20–4,000 ng mL^?1 for danshensu, 50–10,000 ng mL^?1 for SAB, and 10–2,000 ng mL^?1 for HSYA. The method was successfully applied to a pharmacokinetic study of a traditional Chinese medicinal preparation, Danhong lyophilized powder for injection.     

LC–MS/MS Determination of Catecholamines in Urine Using FMOC-Cl Derivatization on Solid-Phase Extraction Cartridge

A method for the determination of catecholamine derivatives in human urine is proposed that includes the derivatization of target compounds on a solid-phase extraction cartridge and determination of the analytes by a UHPLC method with tandem mass-spectrometric detection. 9-Fluorenyl-methoxycarbonyl chloride was used as the derivatization agent. The limits of detection for the analytes were 2.5 ng mL^?1 for 9-fluorenyl-methoxycarbonyl-adrenaline, 5 ng mL^?1 for 9-fluorenyl-methoxycarbonyl-octopamine, and 25 ng mL^?1 for 9-fluorenyl-methoxycarbonyl-dopamine. The proposed procedure was tested on real samples obtained from volunteers.     

Simultaneous Determination of Three Flavonoids in Rat Plasma by RP-LC After Oral Administration of the Total Flavonoids of Scutellaria barbata

A liquid chromatographic method for the simultaneous determination of three flavonoids, scutellarin (SCU), isoscutellarein-8-O-glucuronide (ISO) and luteolin (LUT) in rat plasma was developed and validated. Following a single-step liquid–liquid extraction with ethyl acetate, the analytes and internal standard (IS) (rutin) were successfully separated on a Diamonsil C₁₈ column using a mobile phase composed of acetonitrile (A)–0.2% phosphoric acid aqueous solution (B) (0–5 min, 20% A–29% A; 5–25 min, 29% A, v/v) at a flow rate of 1.0 mL min^?1. The linear range was 0.044–2.20 μg mL^?1 for SCU, 0.042–2.08 μg mL^?1 for ISO, and 0.056–2.80 μg mL^?1 for LUT, with the correlation coefficients of 0.9995, 0.9989 and 0.9963, respectively. The limit of quantification of SCU, ISO and LUT were 44, 41.6 and 56 ng mL^?1, respectively. The accuracy of assay was between 88.4 and 103.0%. The inter-day and intra-day precisions (RSD) were less than 10.5%. The developed method was simple, rapid and applied successfully to study the pharmacokinetics of SCU, ISO and LUT after oral administration of the total flavonoids of Scutellaria barbata.     

LC Determination of Ketorolac in Human Plasma and Urine for Application to Pharmacokinetic Studies

A simple, specific and sensitive liquid chromatographic method has been developed for the assay of ketorolac in human plasma and urine. The clean-up of plasma and urine samples were carried out by protein precipitation procedure and liquid–liquid extraction, respectively. Separation was performed by a Waters sunfire C₁₈ reversed-phase column maintained at 35 °C. The mobile phase was a mixture of 0.02 M phosphate buffer (pH adjusted to 4.5 for plasma samples and to 3.5 for urine samples) and acetonitrile (70:30, v/v) at a flow rate of 1.0 mL min^?1. The UV detector was set at 315 nm. Nevirapine was used as an internal standard in the assay of urine sample. The method was validated over the concentration range of 0.05–8 and 0.1–10 μg mL^?1 for ketorolac in human plasma and urine, respectively. The limits of detection were 0.02 and 0.04 μg mL^?1 for plasma and urine estimation at a signal-to-noise ratio of 3. The limits of quantification were 0.05 and 0.1 μg mL^?1 for plasma and urine, respectively. The extraction recoveries were found to be 99.3 ± 4.2 and 80.3 ± 3.7% for plasma and urine, respectively. The intra-day and inter-day standard deviations were less than 0.5. The method indicated good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy. This assay demonstrated to be applicable for clinical pharmacokinetic studies.     

RP-LC Determination and Pharmacokinetic Study of Ferulic Acid and Isoferulic Acid in Rat Plasma After Taking Traditional Chinese Medicinal-Preparation: Guanxinning Lyophilizer

A sensitive, simple, and accurate method for determination and pharmacokinetic study of ferulic acid and isoferulic acid in rat plasma was developed using a reversed-phase column liquid chromatographic (RP-LC) method with UV detection. Sample preparations were carried out by protein precipitation with the addition of methanol, followed by evaporation to dryness. The resultant residue was then reconstituted in mobile phase and injected into a Kromasil C₁₈ column (250 × 4.6 mm i.d. with 5 μm particle size). The mobile phase was methanol-1% formic acid (33:67, v/v). The calibration plots were linear over the range 5.780–5780 ng·mL^?1 for ferulic acid and 1.740–348.0 ng·mL^?1 for isoferulic acid. Mean recoveries were 85.1% and 91.1%, respectively. The relative standard deviations (RSDs) of within-day and between-day precision were not above 15% for both of the analytes. The limits of quantification were 5.780 ng·mL^?1 for ferulic acid and 1.740 ng·mL^?1 for isoferulic acid. This RP-LC method was used successfully in pharmacokinetic studies of ferulic acid and isoferulic acid in rat plasma after intravenous injection of Guanxinning Lyophilizer.     

Determination of Tangeretin in Rat Plasma by LC-Electrospray-Ion Trap MS

A selective, sensitive, and accurate method has been developed and validated for the quantification of tangeretin in rat plasma. The application of LC-electrospray-ion trap mass spectrometry in full scan and multiple reactions monitoring modes were investigated. Following solid phase extraction using a hydrophilic–lipophilic balance cartridge, the analytes were separated on a C₁₈ column using an isocratic mobile phase composed of acetonitrile/water (50:50, v/v) containing 0.3% formic acid. In full scan mode, the LOQ was 2 ng mL^?1. The standard calibration curve was linear (R ² = 0.9999) over the concentration range 2–200 ng mL^?1. The precision over the concentration range was within 15% (RSD) and the accuracy was ranged from 86 to 115%. In multiple reaction monitoring mode, the LOQ was 1 ng mL^?1 and the standard calibration curve was linear (R ² = 0.9976) over the concentration range 1–100 ng mL^?1 with a precision of 12% and accuracy rangeing from 91 to 113%.     

LC Method for Analysis of Three Flavonols in Rat Plasma and Urine after Oral Administration of Polygonum aviculare Extract

A high-performance liquid chromatographic method has been developed for the simultaneous analysis of the flavonols myricitrin (1), avicularin (2), and juglanin (3) in rat plasma and urine after oral administration of the total flavonoids from Polygonum aviculare. Samples were prepared by solid-phase extraction then separated on a C₁₈ reversed-phase column by use of a mobile-phase gradient prepared from methanol and aqueous formic acid solution. The flow rate was 1 mL min^?1. Detection was performed at 254 nm. The calibration range was 11–1,100 μg mL^?1 for both 2 and 3 in plasma; in urine the calibration ranges for 1, 2, and 3 were 32–1,600, 11–1,100, and 22–1,100 μg mL^?1, respectively. Intra-day and inter-day RSD were less than 4.33 and 3.62% for 2 and 3, respectively, in plasma, and no more than 4.03 and 2.22% for all the analytes in urine. The analytical sensitivity and selectivity of the assay enabled successful application to pharmacokinetic studies of flavonols 1–3 in rats.     

Simultaneous Determination of Amoxicillin and Clavulanic Acid in Dog Plasma by High-Performance Liquid Chromatography Tandem Mass Spectrometry

A rapid, sensitive, and specific high-performance liquid chromatography tandem mass spectrometric method was developed for the simultaneous determination and confirmation of amoxicillin and clavulanic acid in plasma. Plasma sample was subjected to a simple deproteinization with acetonitrile, and then the supernatant was directly diluted by water. Analysis was performed on a Phenomenex Luna C₈ reversed-phase column by detection with mass spectrometry in negative ions multiple reaction monitoring mode. A gradient elution program with 0.1% formic acid and acetonitrile was performed at a flow of 0.25 mL min^?1. There is good linearity in the range of 0.5–500 ng mL^?1 for both amoxicillin and clavulanic acid. The decision limits of amoxicillin and clavulanic acid were 0.06 ng mL^?1 and 0.08 ng mL^?1 in plasma, respectively, and the detection capabilities of two analytes were below 0.5 ng mL^?1. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The extraction recoveries of amoxicillin and clavulanic acid were between 102% and 115% in plasma at three spiked levels of 0.5, 50, and 500 ng mL^?1, with the relative standard deviations less than 15% for each analyte. The developed method was applied to pharmacokinetic studies of amoxicillin and clavulanic acid tablets in healthy beagles.     

Quantification of Teprenone in Human Plasma by HPLC Coupled to Mass Spectrometry: Application to a Bioequivalence Study

A rapid, sensitive, and specific method has been developed for quantification of teprenone (TEP) in human plasma. The analytes were isolated from plasma by liquid–liquid extraction with t-butyl methyl ether. The extracts were analyzed by high-performance liquid chromatography coupled to mass spectrometry (HPLC–MS); gefarnate was used as internal standard (IS). HPLC separation of the analytes was performed on a C₁₈ column with 1:54:45 (v/v) 1% aqueous acetic acid–methanol–acetonitrile as mobile phase; the flow rate was 0.2 mL min^?1. The compounds were ionized by atmospheric-pressure chemical ionization (APCI). Calibration plots for TEP were linear in the range 20.0–2000.0 ng mL^?1; correlation coefficients were >0.9981. The average extraction efficiency for TEP was >67%, method recovery was >95%, the limit of detection (LOD) was 1.0 ng mL^?1, and the intraday and interday coefficients of variation were <7%. This HPLC–MS procedure was used to assess the bioequivalence of TEP tablet and capsule formulations. A single 150-mg dose of each formulation was administered to 18 healthy male volunteers. The study was conducted using an open, randomized, two-period crossover design with a 1-week wash-out interval. Because the 90% CI for C _max and the ratios of the AUCs were all within the 80–125% range stipulated by the US Food and Drug Administration, it was concluded that the TEP tablet and capsule formulations were bioequivalent in terms of rate and extent of absorption.     

Analysis of Isopropyl 3-(3,4-Dihydroxyphenyl)-2-hydroxypropanoate, a Metabolite of Danshensu, from Salvia miltiorrhiza, in Rabbit Plasma

Isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate (IDHP), a metabolite of Danshensu, from Salvia miltiorrhiza, has been proved to have potential as a novel drug for regulation of vasomotor activity in small-resistance vascular circulation. In this presentation we report a new specific method for analysis of IDHP in rabbit plasma. Plasma samples were pretreated with 1.5% formic acid in acetonitrile to remove the protein, and the resulting supernatant was extracted with ethyl acetate. Chromatographic separation was achieved on a C₁₈ column with 15.0% acetonitrile in 0.3% aqueous formic acid (pH 2.2) as mobile phase. Multiple-reaction-mode ion-trap mass spectrometry was selected for accurate analysis of IDHP. The calibration plot was linear in the range 0.1–200.0 ng mL^?1 for plasma samples. The detection limit was 0.02 ng mL^?1. Intra-day and inter-day coefficients of variation were <13.0% and intra-day and inter-day accuracy was within ±8.0% of known concentrations. Finally, the method was used to investigate the pharmacokinetics of IDHP in rabbits; the results indicated IDHP was eliminated rapidly after oral administration.     

Simultaneous Determination of Danshensu and Puerarin in Rat Plasma by LC-MS-MS and Its Application to a Pharmacokinetics and Bioequivalence Study after Oral Administration of Tongmai Dripping Pill and Tongmai Oral Solutions

The active components danshensu (DS) and puerarin (PA) of Tongmai dripping pills (TDP) and oral solution (TOS) were detected in rat plasma after liquid–liquid extraction and oral administration of formulated TDP and TOS. Simultaneous determinations were carried out using electrospray negative ionization mass spectrometry in multiple reaction monitoring mode. The corresponding ion transitions selected for quantitation of DS and PA were at m/z 197.1, 135.0 and m/z 415.2, 294.9, respectively. 3,4-Dihydroxybenzoic acid was used as the internal standard and was monitored at m/z 153.1, 108.9. The linear calibration curves ranged from 9.56 to 637.00 ng mL^?1 and 9.02 to 601.00 ng mL^?1 for DS and PA, respectively. The lowest detectable limit and the lowest quantification limit for both DS and PA in rat plasma were 2.00 and 9.00 ng mL^?1, respectively. The intra-day precision of the assay was less than 10.7% and 8.99% for DS and PA and inter-day precision was less than 14.8% and 14.2% for DS and PA, respectively. The accuracy ranged from 80.56 to 115.3% and 86.91 to 110.6% for DS and PA. This analytical method was applied to a pharmacokinetics and bioequivalence study of DS and PA. Statistical and bioequivalence analyses of DS and PA data for AUC_0–24h and C _max revealed that the 90% confidence intervals for the mean ratio (T/R) of DS and PA for AUC_0–24h and C _max were 91%–106% and 98%–116%, respectively.     

Simultaneous Analysis of Zofenopril and Its Active Metabolite Zofenoprilat in Human Plasma by LC–ESI-MS Using Pre-Column Derivatization with p-Bromophenacyl Bromide

Zofenoprilat is an active metabolite of zofenopril, which is very unstable in plasma because of oxidative degradation of its thiol group. In this method, p-bromophenacyl bromide was used as derivatization reagent, immediately after plasma separation, to react with the free thiol group of zofenoprilat and form the derivative zofenoprilat-p-BPB. After acidification with 50% acetic acid, the derivatized plasma samples were extracted with methyl tert-butyl ether and separated on a C₁₈ column with 40:60 (v/v) 10 mM ammonium acetate buffer solution containing 0.1% formic acid–acetonitrile as mobile phase. Calibration plots were linear over the concentration range 1–500 ng mL^?1 for zofenopril and 2–1,800 ng mL^?1 for zofenoprilat. The method was successfully used to study the bioavailability of zofenopril calcium capsules relative to that of zofenopril calcium tablets in healthy Chinese volunteers.     

Rapid RP-LC Method with Fluorescence Detection for Analysis of Fexofenadine in Human Plasma

A rapid and sensitive reversed-phase high-performance liquid chromatographic method for analysis of fexofenadine in human plasma has been developed and optimized. The analytes were extracted from biological samples by solid-phase extraction on hydrophilic–lipophilic balance cartridges. LC separation was performed on a C₁₈ analytical column (125 mm × 4 mm i.d., 5-μm particles) with 42:58 (v/v) acetonitrile–water adjusted to pH 2.7 with 85% orthophosphoric acid as mobile phase. Fluorescence detection was performed with excitation at 230 nm and emission at 290 nm. The total time for chromatographic separation was 7 min. The method was validated in accordance with EU guidelines by analysis of plasma samples fortified with fexofenadine at concentrations between 0.05 and 800 ng mL^?1. Calibration plots were linear in this range. Mean recovery was typically 94.03% and the detection limit was 0.05 ng mL^?1. The time required for quantitative analysis is shorter than that required by other methods.     

Rapid LC-APCI-MS-MS Method for Simultaneous Determination of Phenacetin and Its Metabolite Paracetamol in Rabbit Plasma

A highly sensitive liquid chromatographic-atmospheric pressure chemical ionization-tandem mass spectrometric method is developed to quantitate phenacetin and its metabolite paracetamol in rabbit plasma. The analytes and internal standard oxazepam are extracted from plasma by liquid–liquid extraction using ethyl acetate, and separated on a Zorbax SB-C₁₈ column (2.1 mm × 150 mm, 5 μm) using acetonitrile–0.1% formic acid in water (40:60 v/v) at a flow of 0.4 mL min^?1. Detection is carried out by multiple reaction monitoring on a ion-trap LC-MS-MS system with an atmospheric pressure chemical ionization interface. The assay is linear over the range 4–1,600 ng mL^?1 for phenacetin and 3–2,000 ng mL^?1 for paracetamol, with a lower limit of quantitation of 4 ng mL^?1 for phenacetin and 3 ng mL^?1 for paracetamol. Intra- and inter-day precision are less than 7.1% and the accuracy are in the range 97.3–103.5%. The validated method is successfully used to analyze the drug in samples of rabbit plasma for pharmacokinetic study.     

Optimization of temperature-controlled ionic liquid homogenous liquid phase microextraction followed by high performance liquid chromatography for analysis of diclofenac and mefenamic acid in urine sample

In this work, a temperature-controlled ionic liquid homogeneous liquid phase microextraction (TCIL-HLPME) technique followed by HPLC–UV was applied for preconcentration and determination of diclofenac (DIC) and mefenamic acid (MEF) in urine samples. 1-butyl-3-methylimidazolium hexafluorophosphate ([C₄mim][PF₆]) was used as the optimum extraction solvent. Experimental design and response surface methodology was used for the optimization process. Firstly, a screening step, using Plackett-Burman design, was carried out to find the significant factors on the extraction efficiency and subsequently, a central composite design (CCD) was employed to find the optimum values of these parameters. The optimal conditions were obtained as extraction solvent volume of 105 µL; sample pH of 2.0, extraction time of 6 min, centrifugation time of 5 min; heating time of 2 min; heating temperature of 50 °C and 20 % of NaCl. Under optimized conditions, the preconcentration factors of 82 and 60 were obtained for DIC and MEF, respectively. The detections limits of 20 and 30 ng mL^?1 were achieved for DIC and MEF by the proposed method, respectively. The calibration curves were linear in the range of 40–1000 and 60–1000 ng mL^?1 for DIC and MEF, respectively. The intra- and inter-assay precisions (RSD %, n = 3) were in the range of 3.5–4.4 % and 7.3–8.0 % at the concentration level of 100 ng mL^?1, respectively. The validated method was successfully applied for the analysis of target analytes in some urine samples.     

An LC Method for the Determination of Bupropion and Its Main Metabolite, Hydroxybupropion in Human Plasma

A new LC method has been developed and validated for the direct determination of bupropion and its main metabolite, hydroxybupropion in human plasma. Plasma samples were analyzed after a simple, one step protein precipitation with trichloroacetic acid using a C₈ column and mobile phase, consisting of methanol/acetonitrile/phosphate buffer (10 mM, pH 3.0) (40:10:50, v/v/v) and 20 mM 1-heptane sulfonic acid sodium salt with carbamazepine as the internal standard. UV detection was performed at 214 and 254 nm. The method was validated over the concentration range of 60–2,400 and 150–4,700 ng mL^?1 for bupropion and hydroxybupropion, respectively. The intra- and inter-day assay variability was less than 15% for the two analytes. Limit of detection values were 24.8 and 63.4 ng mL^?1 for bupropion and hydroxybupropion, respectively. The method developed was applied to quantification of bupropion and hydroxybupropion in human plasma.