Determination of midazolam in rabbit plasma by GC and LC following nasal and ocular administration

GC with nitrogen phosphorus detection and HPLC with UV detection were used to determine midazolam (MDZ) levels in rabbit plasma following ocular and nasal administration. For GC with nitrogen phosphorus detection, the analyte was extracted from the plasma using a three‐step liquid–liquid extraction including extraction with an isopropanol/butyl chloride mixture in an alkaline solution, followed by extractions with 1 M HCl, and finally with an alkaline solution of butyl chloride. The recovery of MDZ was dependent on the sample alkalization time prior to the final extraction. The procedure increased the recovery of MDZ up to 99.6%. Improved sample preparation led to a significant increase in the sensitivity of the determination by GC with nitrogen phosphorus detection. The achieved detection limit was 0.34 ng/mL, which is ten times lower than that obtained using HPLC with UV detection. The small plasma volume was another advantage of the GC with nitrogen phosphorus detection method (200 μL per assay). Both administration routes of the anesthetic (nasal and ocular) resulted in comparable plasma MDZ levels. Kinetic simulation of the MDZ plasma was performed for both administration routes.     

Development and validation of rapid and sensitive LC methods with PDA and fluorescence detection for determination of piribedil in rat plasma and brain tissues and their pharmacokinetic application

Simple, selective and sensitive high‐performance liquid chromatographic (HPLC) bioanalytical methods using fluorescence (FL) and photodiode array (PDA) detectors were developed and validated for determination of piribedil (PBD), an anti‐Parkinson's drug, in rat plasma and brain samples, with telmisartan as internal standard (IS). Protein precipitation technique was used to extract PBD from both biological matrices. Chromatographic separation was achieved on a Phenomenex Kinetex C₁₈ end‐capped column (250 × 4.6 mm, 5 μm), with 38:62 v/v acetonitrile and ammonium acetate buffer (pH 5.0) as mobile phase at 1.0 mL/min flow rate. Linear response in the concentration ranges 5–300 and 150–3000 ng/mL in plasma, and 15–900 and 450–9000 ng/g in brain tissue were achieved in FL and PDA detectors, respectively. The chromatograms were extracted at 239 nm in case of PDA detection and at excitation wavelength of 239 nm and emission wavelength of 385 nm in case of FL detection. FL detection was found to be more sensitive compared with PDA detection. The developed methods were successfully employed in determining the plasma time course, brain distribution and the pharmacokinetic parameters of PBD following intravenous bolus administration of the drug in male Wistar rats.     

Determination of ng/mL levetiracetam using ultra-high-performance liquid chromatography-photodiode absorbance

This paper demonstrates the analysis of levetiracetam, a new chiral antiepileptic drug, at ng/mL levels using an ultra-high-performance liquid chromatography (UHPLC)-photodiode absorbance (PDA) method. Three different sample preparation methods, liquid-liquid extraction with Extrelut, solid phase extraction (SPE) with Oasis HLB and Oasis MAX SPE cartridges, and protein precipitation with organic solvents were carried out. The last preparatory method is the simplest and provides the best recoveries: between 97.1% and 100.4% with RSD value below 5%. The column for separation is BEH C18 column (1.7 μm particle size and 100 × 2.1 mm i.d.) and acetonitrile-phosphate buffer (pH = 6.6; 0.01 M) (10/90 v/v) is the mobile phase. The results obtained are compared to analysis conducted by the HPLC method. The UHPLC method was validated in the range of 2-100 μg/mL levetiracetam concentration (R(2) = 0.9997). LOD and LOQ are 10 ng/mL and 33 ng/mL, respectively. The developed UHPLC method was applied to plasma samples of patient with epilepsy.     

Determination of asymmetric and symmetric dimethylarginines in human plasma by HPLC with electrochemical detection

A new HPLC method was developed and validated for the determination of asymmetric and symmetric dimethylarginines and l ‐arginine in human plasma. After SPE and evaporation of the eluate, the samples were derivatised with an o‐phthaldialdehyde reagent containing 3‐mercaptopropionic acid. The derivatives formed were analysed by isocratic RP‐HPLC with electrochemical detection at +320 mV. The mobile phase consisted of 50 mM phosphate buffer (pH 6.1) containing 10% v/v acetonitrile, the flow rate was 1 mL/min. The retention times of all compounds including monomethylarginine (internal standard) were <24 min. The LODs (S/N 3:1) were 0.012 μM for both dimethylarginines and 0.013 μM for l ‐arginine; the linearity of the method was from 0.1 to 20 μM for both dimethylarginines and from 1 to 200 μM for l ‐arginine. Absolute extraction recoveries measured for all analytes ranged from 85 to 88%.     

Derivative spectrum chromatographic method for the determination of trimethoprim in honey samples using an on-line solid-phase extraction technique

A simple, selective and rapid analytical method for determination of trimethoprim (TMP) in honey samples was developed and validated. This method is based on a SPE technique followed by HPLC with photodiode array detection. After dilution and filtration, aliquots of 500 μL honey samples were directly injected to an on-line SPE HPLC system. TMP was extracted on an RP SPE column, and separated on a hydrophilic interaction chromatography column during HPLC analysis. At the first detection step, the noise level of the photodiode array data was reduced with two-dimensional equalizer filtering, and then the smoothed data were subjected to derivative spectrum chromatography. On the second-derivative chromatogram at 254 nm, the limit of detection and the limit of quantification of TMP in a honey sample were 5 and 10 ng/g, respectively. The proposed method showed high accuracy (60-103%) with adequate sensitivity for TMP monitoring in honey samples.     

高效液相色谱法对水果中多菌灵与福美双残留的同时测定

建立了高效液相色谱同时测定水果中多菌灵和福美双残留量的方法。样品经二氯甲烷提取,OasisHLB固相萃取柱净化后,经C18色谱柱分离,甲醇-0.05 mol/L甲酸铵溶液(60∶40,体积比)洗脱,紫外检测器进行检测。结果表明,多菌灵、福美双的的线性范围分别为0.01~10.0、0.05~10.0 mg/kg,相关系数分别为1.0、0.9999,检出限分别为0.005、0.03 mg/kg,平均加标回收率为80%~109%。该方法成功用于市售水果样品的测定。     

Measurement of Free and Glucuronidated Cardamonin in Rat Plasma and Bile Using UPLC-MS/MS and Its Application to a Pharmacokinetic and Bile Excretion Study

A rapid and accurate ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry(UPLC-MS/MS) method was established and validated for the measurement of two forms of cardamonin, i.e., free and glucuronidated, in the plasma and bile of rats. Cardamonin and an internal standard (1,8-dihydroxyanthraquinone) were extracted from plasma and bile with ethyl ether via liquid-liquid extraction. The analytes in the extracts were separated by an Agilent Zorbax Stable Bond-C₁₈ column(2.1 mm×50 mm, 1.8 μm) under isocratic elution conditions[acetonitrile(A) and 0.1% ammonium formate in water(B), 40:60(volume ratio)] with a flow rate of 0.4 mL/min, and mass spectrometry in negative ion MRM mode was implemented for analysis. Good linearity over the wide ranges of 0.01-5 μg/mL for plasma and 0.025-10 μg/mL for bile samples was acquired. Method validation was performed according to the FDA guidelines for bioanalytical methods.     

Simultaneous determination of desloratadine and pseudoephedrine in human plasma using micro solid-phase extraction tips and aqueous normal-phase liquid chromatography/tandem mass spectrometry

Cation-exchange micro solid-phase extraction (SPE) tips and aqueous normal-phase (ANP) chromatography coupled with tandem mass spectrometry were explored for the rapid, selective and sensitive quantitation of desloratadine and pseudoephedrine in human plasma. A novel micro-SPE device was evaluated for analyte capacity, extraction efficiency and its ability to maximize recovery of an analyte of interest from bioanalytical matrices by successive replicates of linked extraction steps. Ion suppression using two different methods with micro-SPE tips was negligible when compared to protein precipitation. The use of ANP chromatography eliminated the need for sample reconstitution following extraction and was found to be highly selective. A reliable chromatography system was developed with a short duty cycle of 2 min/sample. The proposed bioanalytical method required 50 microL of plasma for the determination of desloratadine and pseudoephedrine at limits of quantitation of 0.1 and 1.25 ng/mL, respectively. The analytical method was validated in accordance with the FDA guidance on bioanalytical method validation; selectivity, linearity, reproducibility and accuracy were all acceptable.     

HPLC analysis of in vivo intestinal absorption and oxidative metabolism of salicylic acid in the rat

In vivo absorption and oxidative metabolism of salicylic acid in rat small intestine was studied by luminal perfusion experiment. Perfusion through the lumen of proximal jejunum with isotonic medium containing 250 μm sodium salicylate was carried out. Absorption of salicylate was measured by a validated HPLC‐DAD method which was evaluated for a number of validation characteristics (specificity, repeatability and intermediate precision, limit of detection, limit of quantification, linearity and accuracy). The method was linear over the concentration range 0.5–50 μg/mL. After liquid–liquid extraction of the perfusion samples oxidative biotransformation of salicylate was also investigated by HPLC‐MS. The method was linear over the concentration range 0.25–5.0 μg/mL. Two hydroxylated metabolites of salicylic acid (2,5‐dihydroxybenzoic acid and 2,3‐dihydroxybenzoic acid) were detected and identified. The mean recovery of extraction was 72.4% for 2,3‐DHB, 72.5% for 2,5‐DHB and 50.1% for salicylic acid, respectively. The methods were successfully applied to investigate jejunal absorption and oxidative metabolism of sodium salicylate in experimental animals. The methods provide analytical background for further metabolic studies of salycilates under modified physiological conditions.     

An HPLC method for the determination of adenosine diphosphate: An important marker of hexokinase activity in metabolic diseases

Hexokinases play a critical role in the cellular uptake and utilization of glucose. As such, they are of fundamental importance to all cells. By catalyzing glucose to produce glucose‐6‐phosphate, hexokinases control the first irreversible step of glucose metabolism and initiate all major pathways of glucose consumption. Our objective was to develop and validate highly sensitive and selective high‐performance liquid chromatography with photodiode array detector (HPLC‐PDA) assays allowing the determination of adenosine diphosphate, which was used for the determination of hexokinase activity. Samples were analyzed by HPLC‐PDA using a C₁₈ analytical column (250 × 4.6 mm) for chromatographic separation. Optimal detection was achieved based on isocratic elution with a mobile phase consisting of a mixture of sodium phosphate monobasic buffer and methanol. This method met all of the requirements of specificity, sensitivity, linearity, precision, accuracy and stability generally accepted in bioanalytical chemistry and was successfully applied to a study of hexokinase activity in an alloxan‐induced diabetic rat model. Determination of hexokinase activity will permit characterization of cellular metabolic state in many diseases, such as cancer and diabetes.     

HPLC determination of (+)-pseudoephedrine and (-)-ephedrine in Japanese herbal medicines containing Ephedra herb using solid-phase extraction

We developed a rapid and simple HPLC method combined with solid-phase extraction (SPE) for quantitative analysis of (+)-pseudoephedrine (PEP) and (-)-ephedrine (EP) in Japanese herbal (Kampo) medicines such as Kakkon-to, Sho-seiryu-to, Goshaku-san and Bofu-tsusho-san. SPE was performed on TOYOPAK IC-SP M containing propylsulfonic groups. Determination of PEP and EP was carried out using ion-pair reversed-phase HPLC with sodium dodecyl sulfate. N-Benzyldiethylamine was used as an internal standard. The analytical procedure was validated with regard to specificity, linearity, accuracy, and precision. These data suggest that the analytical method developed in this study is useful for quantitative analysis of PEP and EP in various formulations of Kampo medicine containing Ephedra herb.     

Isolation and determination of carbendazim residue from wheat grain by matrix solid‐phase dispersion and HPLC

Carbendazim residues were analyzed by column‐switching high‐performance liquid chromatography (HPLC) with photodiode array detection (PDA). The active ingredient was extracted by matrix solid‐phase dispersion (MSPD) from wheat grain on an acidic silica gel column using a methanol‐dichloromethane mixture. The recovery rate for fortified samples was 87.3 ± 3.3% with a standard deviation (SD) of 2.9%. The detection limit was 0.02 μg/mL. The method was applied to the determination of carbendazim residues in wheat grain samples from a treated field.     

HPLC method for the determination and pharmacokinetic studies on geniposide in rat serum after oral administration of traditional Chinese medicinal preparation Yin-Zhi-Ku decoction

A new HPLC method for the determination of geniposide in rat serum with solid-phase extraction (SPE) for preconcentration is described. Geniposide and an internal standard (paeoniflorin) were extracted from serum by SPE using C18 cartridges. Analysis of the extract was then performed on a reversed-phase C18 column using acetonitrile-water (16:84, v/v) as the eluting solvent system, and UV detection at 238 nm was used to measure the analyte with a limit of quantitation about 0.1 microg/mL. The calibration curve for geniposide was linear (r = 0.9993) in the concentration range 0.1-16.0 microg/mL. The intra- and inter-day precision of the geniposide were determined and their RSD did not exceed 10%. The validated method has been successfully applied for pharmacokinetic studies of geniposide from rat serum after oral administration of Yin-Zhi-Ku decoction.     

Analysis of mandipropamid residual levels through systematic method optimization against the matrix complexity of sesame leaves using HPLC/UVD

An analytical method was developed to detect mandipropamid residues in sesame leaves using high‐performance liquid chromatography–ultraviolet detection. Samples were extracted with acetonitrile and were prepurified using a solid‐phase extraction (SPE) cartridge with an additional dispersive‐SPE (d‐SPE) sorbent application. The method was validated using an external calibration curve prepared using pure solvent. The linearity was excellent with determination coefficient = 1. The limits of detection and quantification were 0.003 and 0.01 mg/kg, respectively. Recoveries at three spiking levels – 0.1, 0.5, and 1.0 mg/kg – were in the range 80.3–90.7% with relative standard deviations <2%. This method was applied to field‐treated samples collected from two different areas, Gwangju and Muan, in the Republic of Korea and the half‐lives were similar, 5.10 and 5.41 days, respectively. The pre‐harvest residue limit was also predicted for both sites. The proposed method is sensitive and able to quantify trace amounts of mandipropamid in leafy vegetables. The combination of SPE and d‐SPE effectively removed the matrix components in sesame leaves. Copyright © 2015 John Wiley & Sons, Ltd.     

Gradient HPLC-DAD stability indicating determination of miconazole nitrate and lidocaine hydrochloride in their combined oral gel dosage form

The pharmaceutical combination of miconazole nitrate (MZ) and lidocaine hydrochloride (LD) is used in the curative and prophylactic therapy of the oral and gastro-intestinal infections caused by Candida albicans. To the best of our knowledge, no attempts have yet been made to assay this combination by any analytical method. A simple and selective high-performance liquid chromatography-diode array detection (HPLC-DAD) stability-indicating method was developed for the simultaneous determination of MZ and LD in their combined formulation. Effective chromatographic separation was achieved using a Zorbax SB-C8 column with gradient elution of the mobile phase composed of 0.05 M phosphoric acid and acetonitrile. The gradient elution started with 25% (by volume) acetonitrile, ramped up linearly to 65% in 6 min, then kept constant until the end of the run. The mobile phase was pumped at a flow rate of 1 mL/min. The multiple wavelength detector was set at 215 nm and analytes were quantified by measuring their peak areas. The retention times for LD and MZ were approximately 4.1 and 8.4 min, respectively. The reliability and analytical performance of the proposed HPLC procedure were statistically validated with respect to linearity, ranges, precision, accuracy, selectivity, robustness, detection and quantification limits. Calibration curves were linear in the ranges of 5-100 μg/ml for both drugs with correlation coefficients > 0.999. Both drugs were subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. The proposed method proved to be stability-indicating by the resolution of the two analytes from the related substance and potential impurity (2,6-dimethylaniline) and from the forced-degradation products. The validated HPLC method was applied to the analysis of MZ and LD in the combined oral gel preparation, in which the two analytes were successfully quantified and resolved from the pharmaceutical additives. The proposed method made use of DAD as a tool for peak identity and purity confirmation.     

Systematic evaluation of matrix effect and cross‐talk‐free method for simultaneous determination of zolmitriptan and N‐desmethyl zolmitriptan in human plasma: a sensitive LC‐MS/MS method validation and its application to a clinical pharmacokinetic study

The objective of the present work was to carry out systematic evaluation to eliminate matrix effect owing to plasma phospholipids as observed during sample preparation and to develop a cross‐talk‐free sensitive, selective and rapid bioanalytical method for the simultaneous determination of zolmitriptan (ZT) and N‐desmethyl zolmitriptan (DZT) in human plasma by liquid chromatography–tandem mass spectrometry using naratriptan as internal standard (IS). The analytes and IS were quantitatively extracted from 200 μL human plasma by solid phase extraction. No cross‐talk was found between ZT and DZT having identical product ions. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique, operating in multiple reaction monitoring and positive ion mode. The total chromatographic run time was 2.5 min. The method was fully validated for sensitivity, selectivity, specificity, linearity, accuracy, precision, recovery, matrix effect, dilution integrity and stability studies. The method was validated over a dynamic concentration range of 0.1–15 ng/mL for ZT and DZT. The method was successfully applied to a bioequivalence study of 2.5 mg ZT tablet formulation in 18 healthy Indian male subjects under fasting conditions. Assay reproducibility was assessed by reanalysis of 62 incurred samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Biovalidation of an SPE-HPLC-UV-fluorescence method for the determination of valsartan and its metabolite valeryl-4-hydroxy-valsartan in human plasma

A simple and fast method for the simultaneous determination of the antihypertensive drug Valsartan and its metabolite in human plasma has been validated. The proposed method deals with SPE, followed by an HPLC separation coupled with fluorimetric and photometric detection. The optimization of the SPE-HPLC method was achieved by an experimental design. The separation was performed on an RP C18 Atlantis 100 mmx3.9 mm column. The mobile phase consisted of a mixture of ACN 0.025% TFA and phosphate buffer (5 mM, pH = 2.5) 0.025% TFA and was delivered in gradient mode at a flow rate of 1.30 mL/min. The eluent was monitored with a fluorescence detector at 234 and 378 nm excitation and emission wavelengths, respectively, and at 254 nm using a photometric detector. The full analytical validation was performed according to the Food and Drug Administration (FDA) 'guidance for industry: bioanalytical method validation' and the recoveries obtained for Valsartan and its metabolite ranged from 94.6 to 108.8%. The validated method was successfully applied to 12 plasma samples obtained from patients under antihypertensive treatment with Valsartan.     

Development and validation of a capillary electrophoresis method applied to the analysis of l‐citrulline in an oral formulation for pediatric use

A simple and highly sensitive CE–UV method was applied in the determination of l ‐ctrulline, which was developed from an oral formulation for pediatric use. The novel method was based on the analysis of l ‐citrulline for direct ultraviolet detection at 198 nm. The BGE consisted of 10 mM sodium tetraborate and 50 mM SDS at pH 9, and the electrophoretic parameters were optimized. The method was validated in terms of specificity, linearity, LOD, LOQ, precision, accuracy, and robustness. The LOD and LOQ obtained were 1.36 and 4.54 μg/mL, respectively. In addition, the method offers higher sensitivity and specificity compared with the results obtained from HPLC method using UV‐detectors, in which l‐ citrulline needs to be derivatizated. Furthermore, low cost and simplicity of the system allowed the rapid and simple quantitation of l‐ citrulline in the oral formulation for quality control and stability indicated method.     

HPLC-UV analytical method for determination of pizotifen after in vitro transdermal diffusion studies

Pizotifen malate is an antihistamine and serotonin inhibitor used in the preventive treatment of migraine and eating disorders. A simple, rapid, accurate and precise high‐performance liquid chromatography (HPLC) method involving ultraviolet detection was validated for the quantitative analysis of pizotifen malate in samples from in vitro transdermal diffusion studies. The method was validated for specificity, linearity, accuracy, precision, limit of detection, limit of quantification and robustness. Drug stability in the solution was also determined under different conditions. Separation was carried out using a 250 × 4.0 mm Kromasil^® C₁₈ column at room temperature. The detector response, fitted at 254 nm, was found to be linear in a concentration range between 0.24 and 24.0 µg/mL. The limit of detection was 0.02 µg/mL and the limit of quantification was 0.07 µg/mL. Finally, in vitro transdermal diffusion of pizotifen malate was characterized using the validated HPLC method. Copyright © 2011 John Wiley & Sons, Ltd.     

Development and validation of a reliable high-performance liquid chromatographic method for determination of nodakenin in rat plasma and its application to pharmacokinetic study

A simple and reliable high-performance liquid chromatographic (HPLC) method has been developed for the determination of nodakenin in rat plasma. The concentration of nodakenin was determined in plasma samples after deproteinization with methanol using hesperidin as internal standard. HPLC analysis was performed on a Diamonsil C(18) analytical column using acetonitrile-water (25:75, v/v) as the mobile phase and a UV detection at 330 nm. This method was validated in terms of recovery, linearity, accuracy and precision (intra- and inter-day variation). The extraction recoveries were 91.3 ± 10, 87.8 ± 4.8 and 92.6 ± 5.1 at concentrations of 0.500, 5.00 and 40.0 μg/mL, respectively. The standard curve for nodakenin was linear (r(2) ≥ 0.99) over the concentration range 0.250-50.0 μg/mL with a lower limit of quantification of 0.250 μg/mL. The intra- and inter-day precision (relative standard deviation, RSD) values were not higher than 12% and the accuracy (relative error, RE) was within ± 5.8% at three quality control levels. The validated method was successfully applied for the evaluation of the pharmacokinetics of nodakenin in rats after oral administration of Rhizoma et Radix Notopterygii decoction and nodakenin solution.