Analysis of Insulin by High Performance Liquid Chromatographic Method with Precolumn Derivatization with 4‐Chloro‐7‐Nitrobenzo‐2‐Oxa‐1,3‐Diazole

Abstract

A high performance liquid chromatographic method (HPLC) with precolumn derivatization and fluorescence detection for insulin was developed and applied for the quantification of insulin in spiked serum. To covalence couple with insulin, 4‐chloro‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD‐Cl) was selected as fluorescent reagent. The optimal derivatization conditions were as follows: temperature 50; time 2 h, in the dark; 0.1 M phosphate buffer (pH 9.0). Analytical separation was carried out on a C18 column and the mobile phase including acetonitrile‐water containing 0.1% trifluoroacetic acid (TFA) (v/v∶ 30/70). The excitation/emission wavelengths were 470/540 nm. Under the conditions, the retention time and capacity factor of the adduct of insulin‐NBD were 10.03 min (flow rate 1 mL/min) and 3, respectively. The recovery of insulin in serum was 95.06% and the detection limit was 90 nM. In the investigated concentration ranges (0.46 µM～16.10 µM), R² was 0.9934, which indicated the potential for the application of NBD‐Cl derivatization to the analysis of insulin in the biological matrices, although with the shortcoming of long analytical time.     

Determination of Amantadine Residue in Honey by Solid‐phase Extraction and High‐performance Liquid Chromatography with Pre‐column Derivatization and Fluorometric Detection

Amantadine (AMA) is an anti‐viral drug used in apiculture to protect honeybee against the sacbrood virus (Morator aetatulae). This study described a reliable high‐performance liquid chromatographic (HPLC) method for analyzing AMA in honey using a solid‐phase extraction (SPE) cartridge (Plexa PCX) for purification, 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐F) as a pre‐column derivatization agent, and fluorometric detection (λ_ex=470 nm, λ_em=530 nm). The chromatographic separation was performed on an XDB C18 column (150×4.6 mm i.d.) using 0.1% trifluoroacetic acid/acetonitrile (35:65,V/V) as the mobile phase at a flow rate of 1.0 mL·min⁻¹ with a run time of 20 min. Under these optimal conditions, a linear relationship was observed in the range of 0.025–1.0 µg· mL⁻¹ with a good correlation coefficient (0.998) and low limit of detection (0.0080 µg·g⁻¹), the recoveries were all above 90%, and the intra‐day and inter‐day precision (RSD) ranged from 3.4%–5.1%.     

Simultaneous determination of norisoboldine and its major metabolite in rat plasma by ultra-performance liquid chromatography-mass spectrometry and its application in a pharmacokinetic study

Norisoboldine (NIB) is one of the main bioactive isoquinoline alkaloids in Linderae Radix. A rapid, selective and sensitive method using UPLC‐ESI/MS was first developed for simultaneous determination of NIB and norisoboldine‐9‐O‐α‐glucuronide (NIB‐Glu), its major metabolite in rat plasma. A one‐step protein precipitation with methanol was employed as sample preparation technique. Chromatographic separation was carried out on an Acquity UPLC BEH C₁₈ column (50 × 2.1 mm, i.d. 1.7 µm) with a gradient mobile phase consisting of acetonitrile and water containing 0.1% formic acid. Detection and quantification were performed using a quadrupole mass spectrometer by selective ion reaction‐monitoring mode. Good linearity was achieved using weighted (1/x²) least squares linear regression over the concentration ranges 0.01–2 µg/mL for NIB and 0.025–25 µg/mL for NIB‐Glu. The lower limit of quantification of NIB and NIB‐Glu was 0.01 and 0.025 µg/mL, respectively. The intra‐ and inter‐day precisions (relative standard deviations) of the assay at all three quality control levels were 4.6–14.1% for NIB, and 5.0–12.2% for NIB‐Glu. The accuracies (relative error) were −13.5–8.1% for NIB and −12.8–7.6% for NIB‐Glu, respectively. This developed method was successfully applied to an in vivo pharmacokinetic study in rats after a single intravenous dose of 10 mg/kg NIB. Copyright © 2010 John Wiley & Sons, Ltd.     

HPLC‐FLD determination of NBD‐cholesterol,its ester and other metabolites in cellular lipid extracts

22‐[N(?7‐Nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino]‐23,24‐bisnor‐5‐cholen‐3β‐ol (NBD‐cholesterol), a fluorescent cholesterol analog, was an extragenous cholesterol tracer used to study cholesterol absorption and metabolism in cultured cells. In order to measure free intracellular cholesterol and its esters, a precise and sensitive method employing high‐performance liquid chromatography/fluorescence detection (HPLC‐FLD) was developed for the first time. Method validation showed a limit of detection at 30 ng/mL. The calibration curve was linear within the range of 0.0625–10.0 µg/mL (r² = 0.999). Accuracy and precision were highlighted by good recovery and low variations. Apart from NBD‐cholesteryl oleate, two additional cellular metabolites of NBD‐cholesterol, probably an isomer and an oxidation product, were determined in the lipid extracts of Caco‐2 human colon adenocarcinoma cells according to mass spectrometry. In AC29 mouse malignant mesothelioma cells overexpressing acyl‐CoA:cholesterol acyltransferase‐1 (ACAT1) or ACAT2, only the oxidized metabolite was detected. Using the newly developed method, YIC‐C8‐434, a known ACAT inhibitor, was shown to inhibit ACAT activity in Caco‐2 cells, as well as in AC29/ACAT1 or AC29/ACAT2 cells. In conclusion, the sensitive and specific HPLC‐FLD method is a powerful tool for simultaneous quantification of intracellular NBD‐cholesterol and its oleoyl‐ester. Copyright © 2013 John Wiley & Sons, Ltd.     

A reliable method of liquid chromatography for the quantification of acetaminophen and identification of its toxic metabolite N-acetyl-p-benzoquinoneimine for application in pediatric studies

The aim of the present study was to develop a simple, selective and reliable method to quantify acetaminophen and its toxic metabolite N‐acetyl‐p‐benzoquinoneimine (NAPQI) for pediatric studies using 100 µL plasma samples, by reverse‐phase HPLC and UV detection. The assay was performed using a C₁₈ column and an isocratic elution with water–methanol–formic acid (70:30:0.15; v/v/v) as mobile phase. Linearity of the method was assayed in the range of 1–30 µg/mL for acetaminophen and 10–200 µg/mL for NAPQI, with a correlation coefficient r = 0.999 for both compounds, and inter‐ and intra‐day coefficients of variation of less than 13%. Several commonly co‐administered drugs were analyzed for selectivity and no interference with the determinations was observed. The detection and quantification limits for acetaminophen and NAPQI were 0.1 and 1 µg/mL, and 0.1 and 10 µg/mL respectively. The present method can be used to monitor acetaminophen levels using 100 µL plasma samples, which may be helpful when very small samples need to be analyzed, as in pharmacokinetics determination or drug monitoring in plasma in children. This assay is also able to detect the NAPQI for drug monitoring in patients diagnosed with acetaminophen intoxication. Copyright © 2010 John Wiley & Sons, Ltd.     

Liquid Chromatographic Analysis of Cephalexin in Human Plasma by Fluorescence Detection of the 9-Fluorenylmethyl Chloroformate Derivative

Abstract

A simple and sensitive precolumn derivatization method for the determination of cephalexin in human plasma has been developed. Cephalexin was derived with 9-fluorenylmethyl chloroformate (FMOC-Cl) in borate buffer (5 mM, pH 8.5) for 15 min at 25°C. Optimal conditions for the derivatization were described. The derivative was chromatographed on an XDB-C₁₈ column with water–acetonitrile (10:90, v/v) as mobile phase at a flow rate of 1.0 mL/min. The fluorescence excitation and emission wavelengths were 268 nm and 314 nm, respectively. The standard curve in spiked plasma was linear over the range of 0.0234–58.5 µg/mL; the detection limit (signal-to-noise ratio = 3; injection volume, 10 µL) was about 0.014 µg/mL. The performance of analysis was studied, and the validated method showed excellent performance in terms of selectivity, sensitivity, precision, and accuracy.     

Development of a method for the determination of cefovecin in plasma by HPLC

A simple high‐performance liquid chromatography method for the determination of cefovecin in small volume plasma has been developed. Following solid‐phase extraction using Oasis HLB cartridges, samples were separated by reverse‐phase high‐performance liquid chromatography on an XBridge C₈ (3.5 µm) 4.6 × 250 mm column and quantified using ultraviolet detection at 280 nm. The mobile phase was a mixture of 10 mm ammonium acetate (pH 3.5) and acetonitrile (89:11), with a flow rate of 0.85 mL/min. The standard curve ranged from 0.1 to 200 µg/mL. Intra‐ and Inter‐assay variability for cefovecin was <10%, and the average recovery was >90%. The lower limit of quantitation was 0.1 µg/mL. This method was successfully applied to the analysis of cefovecin samples at our institution. This is also the first fully validated method with an internal standard that does not use mass spectrometry. Copyright © 2014 John Wiley & Sons, Ltd.     

Liquid Chromatographic Determination of Glyoxal and Methylglyoxal from Serum of Diabetic Patients using Meso‐Stilbenediamine as Derivatizing Reagent

Abstract

Meso‐stilbenediamine has been used as derivatizing reagent for liquid chromatographic (LC) determination of glyoxal (Go), methylglyoxal (MGo), and dimethylglyoxal (DMGo) at pH 3. Liquid chromatographic elution and separation was carried out from the column Kromasil 100 C‐18, 5 µm (15×0.46 mm i.d.) with methanol: water:acetonitrile (59:40:1, v/v/v) with a flow rate of 1 mL/min and ultraviolet detection at 254 nm. The linear calibration curves were obtained for Go, MGo, and DMGo within 0.97–4.86 µg/mL, 1.52–7.6 µg/mL, and 1.41–7.08 µg/mL with detection limits of 48 ng/mL, 76 ng/mL, and 70.8 ng/mL, respectively. The method was applied for the determination of Go and MGo from serum of patients suffering from diabetes and ketosis. The amounts of Go and MGo found were 0.150–0.260 µg/mL and 0.160–0.270 µg/mL with coefficient of variation (C.V.) 2.6–4.7% and 2.5–4.6%, respectively. The results obtained were compared with normal subjects with Go and MGo contents of 0.025–0.065 µg/mL and 0.030–0.070 µg/mL with C.V 1.5–4.9% and 1.6–4.8% in the serum.     

Development and validation of a RP-HPLC method for simultaneous quantification of bedaquiline (TMC207), moxifloxacin and pyrazinamide in a pharmaceutical powder formulation for inhalation

A reversed phase high performance liquid chromatography (RP-HPLC) method for the simultaneous quantification of bedaquiline (TMC207), moxifloxacin and pyrazinamide in a pharmaceutical powder formulation for inhalation has been developed and validated. The powder was simply dissolved in methanol and the analytes separated in a run time of 20?min on a Luna C18 (2) (150?×?4.6?mm, 5?µm) column using gradient elution with methanol and triethylamine phosphate buffer (pH 2.5) delivered at 1.2?mL/min. The detection (with retention time) was carried out at 269?nm (2.9?min) for pyrazinamide, 296?nm (7.0?min) for moxifloxacin and 225?nm (16.3?min) for bedaquiline, respectively. The method was linear for all analytes in the concentration range 1-100?µg/mL with correlation coefficients >0.998. Lower limits of quantitation (µg/mL) of bedaquiline, moxifloxacin and pyrazinamide were 0.56, 0.43 and 0.24, respectively. The method was accurate (relative error in the range ?0.2 to 2.2) and precise (%RSD ≤6.2) with recovery in the range 100.0–104.7%. The method was successfully applied to determine the drug content and content uniformity of the three analytes in a spray-dried combination powder formulation for inhalation containing L-leucine.     

A simple and sensitive assay for eflornithine quantification in rat brain using pre‐column derivatization and UPLC‐MS/MS detection

Eflornithine (α‐difluoromethylornithine) has been used to treat second‐stage (or meningoencephalitic‐stage) human African trypanosomiasis and currently is under clinical development for cancer prevention. In this study, a new ultraperformance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS)‐based assay was developed and validated for the quantification of eflornithine in rat brain. To improve chromatographic retention and MS detection, eflornithine was derivatized with 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate for 5 min at room temperature prior to injection. Derivatized eflornithine was separated on a reverse‐phase C₁₈ UPLC column with a 6‐min gradient; elution occurred at approximately 1.5 min. Prior to derivatization, eflornithine was reproducibly extracted from rat brain homogenate by methanol protein precipitation (~70% recovery). Derivatized eflornithine was stable in the autosampler (6 °C) for at least 24 h. This new assay had acceptable intra‐ and interday accuracy and precision over a wide dynamic range (5000‐fold) and excellent sensitivity with a lower limit of quantification of 0.1 µm (18 ng/mL) using only 10 μL of rat brain homogenate. The validated eflornithine assay was applied successfully to determine eflornithine distribution in different regions of rat brain in an in situ rat brain perfusion study. Copyright © 2014 John Wiley & Sons, Ltd.     

Development of a liquid chromatography assay for the determination of opicapone and BIA 9–1079 in rat matrices

Opicapone is a novel potent, reversible and purely peripheral third generation catechol‐O‐methyltransferase inhibitor, currently under clinical trials as an adjunct to levodopa therapy for Parkinson's disease. To support additional nonclinical pharmacokinetic studies, a novel high‐performance liquid chromatographic method coupled to a diode array detector (HPLC‐DAD) to quantify opicapone and its active metabolite (BIA 9–1079) in rat plasma and tissues (liver and kidney) is herein reported. The analytes were extracted from rat samples through a deproteinization followed by liquid‐liquid extraction. Chromatographic separation was achieved in less than 10 min on a reversed‐phase C₁₈ column, applying a gradient elution program with 0.05 M monosodium phosphate solution (pH 2.45 ± 0.05) and acetonitrile. Calibration curves were linear (r² ≥ 0.994) within the ranges of 0.04‐6.0 µg/mL for both analytes in plasma, 0.04‐4.0 µg/mL for opicapone in liver and kidney homogenates, and 0.07‐4.0 µg/mL and 0.06‐4.0 µg/mL for BIA 9–1079 in liver and kidney homogenates, respectively. The overall intra‐ and inter‐day accuracy ranged from ?12.68% to 7.70% and the imprecision values did not exceed 11.95%. This new HPLC‐DAD assay was also successfully applied to quantify opicapone and BIA 9–1079 in a preliminary pharmacokinetic study. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of levetiracetam in human plasma by online heart‐cutting liquid chromatography: Application to therapeutic drug monitoring

Levetiracetam is an antiepileptic drug for the treatment of psychiatric patients. In this study, a selective, straightforward, and rapid online heart‐cutting liquid chromatography method was developed for the therapeutic drug monitoring of levetiracetam. This method allows for the determination of levetiracetam in human plasma without complex sample preparation. The mobile phases consisted of 30 mM aq. orthophosphoric acid solution/methanol (70:30) at a flow rate of 1 mL/min for the first system and 10 mM aq. orthophosphoric acid solution/methanol (55:45) at a flow rate of 1 mL/min for the second system. The first separation was carried out on a GL Sciences Intersil ODS‐3 column (4.6 mm × 150 mm, 3 µm) and the second separation was carried out on a Restek Ultra PFPP column (4.6 mm × 150 mm, 5 µm). The detection was carried out at 205 nm for both systems. The method was validated for selectivity and linearity, which were in the 6–60 µg/mL range. Intra‐ and interassay accuracies were <112.6%, and the intra‐ and interassay precisions were <6.4% for all quality control samples. The lower limit of quantitation was 6 µg/mL. The developed method was successfully applied for therapeutic drug monitoring of plasma samples from patients.     

Determination of a novel nonfluorinated quinolone,nemonoxacin, in human feces and its glucuronide conjugate in human urine and feces by high‐performance liquid chromatography–triple quadrupole mass spectrometry

Three methods were developed and validated for determination of nemonoxacin in human feces and its major metabolite, nemonoxacin acyl‐β‐ d ‐glucuronide, in human urine and feces. Nemonoxacin was extracted by liquid–liquid extraction in feces homogenate samples and nemonoxacin acyl‐β‐ d ‐glucuronide by a solid‐phase extraction procedure for pretreatment of both urine and feces homogenate sample. Separation was performed on a C₁₈ reversed‐phase column under isocratic elution with the mobile phase consisting of acetonitrile and 0.1% formic acid. Both analytes were determined by liquid chromatography–tandem mass spectrometry with positive electrospray ionization in selected reaction monitoring mode and gatifloxacin as the internal standard. The lower limit of quantitation (LLOQ) of nemonoxacin in feces was 0.12 µg/g and the calibration curve was linear in the concentration range of 0.12–48.00 µg/g. The LLOQ of the metabolite was 0.0010 µg/mL and 0.03 µg/g in urine and feces matrices, while the linear range was 0.0010–0.2000 µg/mL and 0.03–3.00 µg/g, respectively. Validation included selectivity, accuracy, precision, linearity, recovery, matrix effect, carryover, dilution integrity and stability, indicating that the methods can quantify the corresponding analytes with excellent reliability. The validated methods were successfully applied to an absolute bioavailability clinical study of nemonoxacin malate capsule. Copyright © 2014 John Wiley & Sons, Ltd.     

Stereospecific high‐performance liquid chromatography of taxifolin,applications in pharmacokinetics,and determination in tu fu ling (Rhizoma smilacis glabrae) and apple (Malus × domestica)

A stereospecific method of analysis of racemic taxifolin (+/?3,5,7,3′,4′‐pentahydroxyflavanone) in biological fluids is necessary to study pharmacokinetics and disposition in fruit and herbs. A simple high‐performance liquid chromatographic method was developed for the determination of all four taxifolin enantiomers. Separation was achieved on a Chiralcel® OJ‐RH column with UV detection at 288 nm. The standard curves in serum were linear over a range of 0.5–100.0 µg/mL for each enantiomer. The mean extraction efficiency was >88.0%. Precision of the assay was <15% (CV), and was within 12% at the limit of quantitation (0.5 µg/mL). The bias of the assay was <15%, and was within 6% at the limit of quantitation. The assay was successfully applied to stereospecific disposition of taxifolin enantiomers in rats and to the quantification of taxifolin enantiomers in tu fu ling (Rhizoma smilacis glabrae) and apple (Malus × domestica). Copyright © 2009 John Wiley & Sons, Ltd.     

A high‐performance liquid chromatographic analysis and preliminary pharmacokinetic characterization of 3′‐hydroxypterostilbene in rats

A high‐performance liquid chromatographic method was developed for the analysis of 3'‐hydroxypterostilbene. This method involves the use of a Luna^® C₁₈ column with ultraviolet detection at 325 nm. The mobile phase consisted of acetonitrile, water and formic acid (50:50:0.01, v/v/v) with a flow rate of 0.8 mL/min. The calibration curves were linear over the range 0.5–100.0 µg/mL. The mean extraction efficiency was between 97.40 and 111.16%. The precision of the assay was 0.196–14.39% (RSD%), and within 15% at the limit of quantitation (0.5 µg/mL). The bias of the assay was <16% and within 15% at the limit of quantitation. This assay was successfully applied to pre‐clinical pharmacokinetic samples from rat urine and serum. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of Cefalothin and Cefazolin in Human Plasma,Urine and Peritoneal Dialysate by UHPLC‐MS/MS: application to a pilot pharmacokinetic study in humans

An ultra‐high‐performance liquid chromatography–tandem mass spectrometry (UHPLC‐MS/MS) method for the analysis of cefazolin and cefalothin in human plasma (total and unbound), urine and peritoneal dialysate has been developed and validated. Total plasma concentrations are measured following protein precipitation and are suitable for the concentration range of 1–500 µg/mL. Unbound concentrations are measured from ultra‐filtered plasma acquired using Centrifree^® devices and are suitable for the concentration range of 0.1–500 µg/mL for cefazolin and 1–500 µg/mL for cefalothin. The urine method is suitable for a concentration range of 0.1–20 mg/mL for cefazolin and 0.2–20 mg/mL for cefalothin. Peritoneal dialysate concentrations are measured using direct injection, and are suitable for the concentration range of 0.2–100 µg/mL for both cefazolin and cefalothin. The cefazolin and cefalothin plasma (total and unbound), urine and peritoneal dialysate results are reported for recovery, inter‐assay precision and accuracy, and the lower limit of quantification, linearity, stability and matrix effects, with all results meeting acceptance criteria. The method was used successfully in a pilot pharmacokinetic study with patients with peritoneal dialysis‐associated peritonitis, receiving either intraperitoneal cefazolin or cefalothin. Copyright © 2015 John Wiley & Sons, Ltd.     

Validated LC‐MS method for simultaneous quantitation of catalpol and harpagide in rat plasma: application to a comparative pharmacokinetic study in normal and diabetic rats after oral administration of Zeng‐Ye‐Decoction

A simple and efficient liquid chromatography‐mass spectrometry (LC‐MS) method was developed and validated for simultaneous quantitation of catalpol and harpagide in normal and diabetic rat plasma. Protein precipitation extraction with acetonitrile was carried out using salidroside as the internal standard (IS). The LC separation was performed on an Elite C₁₈ column (150 × 4.6 mm, 5 µm) with the mobile phase consisting of acetonitrile and water within a runtime of 12.0 min. The analytes were detected without endogenous interference in the selected ion monitoring mode with positive electrospray ionization. Calibration curves offered satisfactory linearity (r > 0.99) at linear range of 0.05–50.0 µg/mL for catalpol and 0.025–5.0 µg/mL for harpagide with the lower limits of quantitation of 0.05 and 0.025 µg/mL, respectively. Intra‐ and inter‐day precisions (RSD) were <9.4%, and accuracy (RE) was in the ?6.6 to 4.9% range. The extraction efficiencies of catalpol, harpagide and IS were all >76.5% and the matrix effects of the analytes ranged from 86.5 to 106.0%. The method was successfully applied to the pharmacokinetic study of catalpol and harpagide after oral administration of Zeng‐Ye‐Decoction to normal and diabetic rats, respectively. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of amino acid neurotransmitters in rat hippocampi by HPLC‐UV using NBD‐F as a derivative

A simple, rapid and accurate high‐performance liquid chromatography method with ultraviolet–visible detection was developed for the determination of five amino acid neurotransmitters – aspartate, glutamic acid, glycine, taurine and γ‐aminobutyric acid – in rat hippocampi with pre‐column derivatization with 4‐fluoro‐7‐nitrobenzofurazan. Several conditions which influenced derivatization and separation, such as pH, temperature, acetonitrile percentage mobile phase and flow rate, were optimized to obtain a suitable protocol for amino acids quantification in samples. The separation of the five neurotransmitter derivatives was performed on a C₁₈ column using a mobile phase consisting of phosphate buffer (0.02 mol/L, pH 6.0)–acetonitrile (84:16, v/v) at a flow rate of 1.0 mL/min with the column temperature at 30°C. The detection wavelength was 472 nm. Without gradient elution, the five neurotransmitter derivatives were completely separated within 15 min. The linear relation was good in the range from 0.50 to 500 µmol/L, and the correlation coefficients were ≥0.999. Intra‐day precision was between 1.8 and 3.2%, and inter‐day precision was between 2.4 and 4.7%. The limits of detection (signal‐to‐noise ratio 3) were from 0.02 to 0.15 µmol/L. The established method was used to determine amino acid neurotransmitters in rat hippocampi with satisfactory recoveries varying from 94.9 to 105.2%. Copyright © 2013 John Wiley & Sons, Ltd.     

A validated HPLC‐PDA method for identification and quantification of two bioactive alkaloids,ephedrine and cryptolepine,in different Sida species

A simple, rapid, accurate and reproducible reverse‐phase HPLC method has been developed for the identification and quantification of two alkaloids ephedrine and cryptolepine in different extracts of Sida species using photodiode array detection. Baseline separation of the two alkaloids was achieved on a Waters RP‐18 X‐terra column (250 × 4.6 mm, 5 µm) using a solvent system consisting of a mixture of water containing 0.1% Trifluoroacetic acid (TFA) and acetonitrile in a gradient elution mode with detection at 210 and 280 nm for ephedrine and cryptolepine, respectively. The calibration curves were linear in a concentration range of 10–250 µg/mL for both the alkaloids with correlation coefficient values >0.99. The limits of detection and quantification for ephedrine and cryptolepine were 5 and 10 µg/mL and 2.5 and 5 µg/mL, respectively. Relative standard deviation values for intra‐day and inter‐day precision were 1.22 and 1.04% for ephedrine and 1.71 and 2.06% for cryptolepine, respectively. Analytical recovery ranged from 92.46 to 103.95%. The developed HPLC method was applied to identify and quantify ephedrine and cryptolepine in different extracts of Sida species. Copyright © 2013 John Wiley & Sons, Ltd.     

High-performance liquid chromatographic determination of memantine hydrochloride in rat plasma using sensitive fluorometric derivatization

In this study, we investigated a simple, sensitive and reliable liquid chromatography‐fluorescence detection method for the determination of memantine hydrochloride in rat plasma which was based on derivatization with 9‐fluorenylmethyl chloroformate (FMOC‐Cl). For the first time, FMOC‐Cl was introduced into derivatization of memantine hydrochloride in rat plasma. The amino groups of memantine hydrochloride and amantadine hydrochloride (internal standard) were trapped with FMOC‐Cl to form memantine hydrochloride‐FMOC‐Cl and amantadine hydrochloride‐FMOC‐Cl compositions, which can be very compatible for LC‐FLD. Precipitation of plasma proteins by acetonitrile was followed by vortex mixing and centrifugation. Chromatographic separation was performed on a C₁₈ column (DIAMONSIL 150×4.6 mm, id 5 μm) with a mobile phase consisting of acetonitrile and water at a flow rate of 1.0 mL/min. The retention times of memantine hydrochloride‐FMOC‐Cl and amantadine hydrochloride‐FMOC‐Cl compositions were 23.69 and 40.27 min, respectively. Optimal conditions for the derivatization of memantine hydrochloride were also described. The limit of quantification (LOQ) was 25 ng/mL for memantine hydrochloride in plasma, the linear range was 0.025–5.0 μg/mL in plasma with a correlation coefficient (r) of 0.9999. The relative standard deviations (RSDs) of intra‐day and inter‐day assays were 4.46–12.19 and 5.23–11.50%, respectively. The validated method was successfully applied to the determination of memantine hydrochloride in rat plasma samples.