Development and Validation of a Stability-Indicating LC Method for Simultaneous Analysis of Aceclofenac and Paracetamol in Conventional Tablets and in Microsphere Formulations

A stability-indicating reversed-phase LC method for analysis of aceclofenac and paracetamol in tablets and in microsphere formulations has been developed and validated. The mobile phase was 80:20 (v/v) methanol–phosphate buffer (10 mM at pH 2.5 ± 0.02). UV detection was at 276 nm. The method was linear over the concentration ranges 16–24 and 80–120 μg mL^?1 for aceclofenac and paracetamol, respectively, with recovery in the range 100.9–102.22%. The limits of detection and quantitation for ACF were 0.0369 and 0.1120 μg mL^?1, respectively; those for PCM were 0.0631 and 0.1911 μg mL^?1, respectively.     

Simultaneous Determination of Ibuprofen and Diphenhydramine Citrate in Tablets by Validated LC

A stability-indicating LC method was developed for the simultaneous determination of ibuprofen and diphenhydramine citrate in pharmaceutical dosage forms. The chromatographic separation was achieved on an Inertsil ODS 3V, 150 × 4.6 mm, 5 μm, column. The mobile phase contained a mixture of 50 mM potassium dihydrogen phosphate buffer:acetonitrile:triethylamine:glacial acetic acid (55:45:0.2:0.2, v/v/v/v). This method allowed the determination of 2.85–9.14 mg mL^?1 of ibuprofen and 0.54–1.73 mg mL^?1 of diphenhydramine citrate, in a diluent consisting of pH 7.2, 50 mM potassium dihydrogen phosphate buffer:acetonitrile (40:60, v/v). The flow rate was 1.2 mL min^?1 and the detection wavelength was 260 nm. The limit of detection for ibuprofen and diphenhydramine citrate was 1.72 and 0.54 μg mL^?1 and the limit of quantification was 5.73 and 1.64 μg mL^?1, respectively. This method was validated for accuracy, precision and linearity. The method was also found to be stability indicating.     

Simultaneous Determination of Acetaminophen, Caffeine, and Chlorphenamine Maleate in Paracetamol and Chlorphenamine Maleate Granules

A simple and rapid HPLC method using phenacetin (PHN) as internal standard has been developed for simultaneous determination of acetaminophen, caffeine, and chlorphenamine maleate in the product compound paracetamol and chlorphenamine maleate granules. Separation and quantitation were achieved on a 250 mm × 4.6 mm, 5 μm particle, C₁₈ column. The mobile phase was methanol 0.05 mol L^?1 aqueous KH₂PO₄ solution, 45:55 (v/v), containing 0.1% triethylamine and adjusted to pH 3.6 by addition of phosphoric acid; the flow rate was 1.0 mL min^?1. Detection of all compounds was by UV absorbance at 260 nm and elution of the analytes was achieved in less than 12 min. The linearity, accuracy, and precision of the method were acceptable to good over the concentration ranges 6.4–153.6 μg mL^?1 for acetaminophen, 5.0–120.0 μg mL^?1 for caffeine, and 9.6–230.4 μg mL^?1 for chlorphenamine maleate.     

Development and Validation of a Stability-Indicating LC Method for Curcumin

A simple, isocratic, stability-indicating liquid chromatographic method for quantitative determination of curcumin was successfully developed. The chromatographic separations were achieved using a Hi-Q-Sil C18; 4.6 mm × 250 mm and 10 μm particle size column employing acetonitrile and acetate buffer (pH 3.0; 60: 40, v/v) as the mobile phase. The analyte was subjected to acidic, basic, oxidative, thermal and photo degradation. The method was validated with respect to linearity, precision, accuracy, limit of detection and limit of quantification. Curcumin was detected by UV-Vis detector at 425 nm whereas the degradation products were detected at 280 nm. The method was linear over the concentration range of 1–10 μg mL^?1. The limit of detection was found to be 0.06 μg mL^?1 and the quantification limit was 0.21 μg mL^?1. Considerable degradation of the analyte was observed when it was subjected to alkaline conditions. Accuracy, evaluated as recovery, was in the range of 97–103%. Intra-day precision and intermediate precision showed relative standard deviations <1% and <2% respectively.     

Simultaneous Determination of Paracetamol and Caffeine in Human Plasma by LC–ESI–MS

A rapid, selective and convenient liquid chromatography–mass spectrometric method for the simultaneous determination of paracetamol and caffeine in human plasma was developed and validated. Analytes and theophylline [internal standard (I.S.)] were extracted from plasma samples with diethyl ether-dichloromethane (3:2, v/v) and separated on a C₁₈ column (150 × 4.6 mm ID, 5 μm particle size, 100 Å pore size). The mobile phase consisted of 0.2% formic acid–methanol (60:40, v/v). The assay was linear in the concentration range between 0.05 and 25 μg mL^?1 for paracetamol and 10–5,000 ng mL^?1 for caffeine, with the lower limit of quantification of 0.05 μg mL^?1 and 10 ng mL^?1, respectively. The intra- and inter-day precision for both drugs was less than 8.1%, and the accuracy was within ±6.5%. The single chromatographic analysis of plasma samples was achieved within 4.5 min. This validated method was successfully applied to study the pharmacokinetics of paracetamol and caffeine in human plasma.     

A Stability-Indicating Assay of Brimonidine Tartrate Ophthalmic Solution and Stress Testing Using HILIC

A stability-indicating hydrophilic interaction liquid chromatography (HILIC) method has been developed and validated for the quantitative determination of Brimonidine tartrate (BT) formulated as an ophthalmic solution. Isocratic separation was achieved using an acetonitrile-buffer mixture (92:8, v/v) at pH 7.1 on an unmodified silica column (250 × 4.6 mm, 5 μm). The drug was subjected to oxidative, hydrolytic, photolytic and thermal stress conditions and complete separation was achieved for the parent compound and degradation products. The influence of acetonitrile, pH and ionic strength of the buffer was studied. Linearity range and recoveries for BT were 100–400 μg mL^?1 and 100.12%, respectively. The method was validated for BT and indicated that the method was sufficiently sensitive with a limit of detection at 0.005 μg mL^?1 and a limit of quantitation at 0.02 μg mL^?1, respectively.     

LC Determination and Pharmacokinetic Study of Gemfibrozil in Human Plasma

A simple and sensitive LC method for the quantitative determination of gemfibrozil in human plasma samples is described. Mometasone furoate was used as the internal standard. Plasma samples were pretreated by protein precipitation using methanol. Separation was performed at 40 °C on a YMC^® ODS-A reverse phase column (5 μm particle size, 150 mm × 4.6 mm i.d.) using 0.2% (v/v) triethylamine in water (adjusting to pH 4.0 with phosphoric acid) and acetonitrile (45:55, v/v) as mobile phase which was delivered at 1.5 mL min^?1. Ultraviolet detection was performed at 230 nm. The linear concentration range for gemfibrozil was 0.25–50 μg mL^?1. The detection limit of this method was 0.1 μg mL^?1. Intra- and inter-assay RSD ranged from 0.63 to 2.04% and 1.37 to 4.27%, respectively. The method was sensitive, simple and repeatable enough to be used in pharmacokinetic studies.     

Determination of Dutasteride by LC: Validation and Application of the Method

A simple, rapid, and stability-indicating reversed-phase high-performance liquid chromatographic (LC) method for analysis for dutasteride has been successfully developed. Chromatography was performed on a 150 mm × 4.6 mm C₁₈ column with acetonitrile–water 60:40 (v/v) as isocratic mobile phase at 1.0 mL min^?1. Ultraviolet detection of dutasteride was at 210 nm. Its retention time was approximately 10 min and its peak was symmetrical. Response was a linear function of concentration over the range 0.2–1 μg mL^?1 (R ² = 0.997) and the limits of detection and quantitation were was 0.05 and 0.10 μg mL^?1, respectively. The method was validated for linearity, precision, repeatability, sensitivity, and selectivity. Selectivity was validated by subjecting dutasteride stock solution to photolytic, acidic, basic, oxidative, and thermal degradation. The peaks from the degradation products did not interfere with that from dutasteride. The method was used to quantify dutasteride in pharmaceutical preparations.     

LC Simultaneous Determination of Thioctic Acid, Benfotiamine and Cyanocobalamin in Thiotacid Compound Capsules

A simple, selective, sensitive, precise, simultaneous liquid chromatographic analysis of capsules containing thioctic acid, benfotiamine and cyanocobalamin was described. Good chromatographic separation was achieved using a Zorbax C18 (4.6 cm × 250 mm, 5 μm) and a mobile phase consisting of acetonitrile–phosphate buffer pH 3.5 (15:85, v/v) at a flow rate of 0.9 mL min^?1. The ultraviolet detector was set a wavelength of 280 nm. Thioctic acid, benfotiamine and cyanocobalamin were eluted at 2.869, 3.752 and 13.689 min, respectively. The linear ranges for thioctic acid, benfotiamine and cyanocobalamin were 30–180, 4–24 and 0.025–0.150 μg mL^?1, respectively. The recoveries of thioctic acid, benfotiamine and cyanocobalamine in pharmaceutical preparation were all greater than 98% and their relative standard deviations were less than 2.0%. The limits of detection were 2.57, 0.19 and 0.003 μg mL^?1 for thioctic acid, benfotiamine and cyanocobalamin, respectively.     

LC Analysis of Isepamicin in Plasma Samples Post-Inhalation with Fluorescence Detection and Its Application to a Pharmacokinetic Study

A simple and novel LC method has been developed for determination of isepamicin (ISP) in rat plasma, an aminoglycoside antibiotic agent. After protein precipitation and clean-up procedure to remove lipophilic contaminants, ISP is derivatized by pre-column with 9-fluorenylmethyl chloroformate for fluorescence detection. Chromatographic separations are achieved using a C18 column and mobile phase consisting of water and acetonitrile (68/32, v/v). Amikacin was used as an internal standard. The calibration curve was linear over a concentration range of 0.625–15 μg mL^?1. The limit of quantification was 0.45 μg mL^?1. The intra- and inter-day variabilities of ISP were both less than 5%. Both derivatives were stable for at least a week at ambient condition. This assay procedure should have useful application in therapeutic drug monitoring of ISP. The limit of detection was 0.10 μg mL^?1. The specificity, assay linearity, low level assay linearity and assay repeatability were also investigated. The established method provides a reliable bioanalytical method to carry out isepamicin pharmacokinetics in rat plasma.     

Gas Chromatographic Determination of Guanidino Compounds Using Hexafluoroacetylacetone and Ethyl Chloroformate as Derivatizing Reagents

Guanidino compounds guanidine, methylguanidine, guanidinoacetic acid, guanidinobutyric acid, guanidinopropionic acid, and guanidinosuccinic acid after derivatization with hexafluoroacetylacetone and ethyl chloroformate at pH 9 in aqueous phase, eluted, and separated from gas chromatographic column HP-5 (30 m × 0.32 mm id) with film thickness of 0.25 μm at an initial column temperature 90 °C for 2 min, followed by heating rate of 10 °C min^?1 up to 220 °C with nitrogen flow rate of 1 mL min^?1. The detection was by flame ionization detector. The linear calibration ranges of each of guanidino compounds were obtained within 1–10 μg mL^?1, and the limit of detection was within 0.014–0.19 μg mL^?1. The derivatization and gas chromatography elution and separation were repeatable in terms of retention time and peak height/peak area with relative standard deviation (RSD) (n = 4) within 1.7–2.9 % and 1.4–2.8 %, respectively. The method was applied for the determination of guanidino compounds from deproteinized serum of uremic patients and healthy volunteers, and was found in the range below the limit of quantitation (BLOQ) to 1.25 μg mL^?1 with RSD within 1.4–3.6 %, and BLOQ to 0.4 μg mL^?1 with RSD 1.3–3.4 %, respectively. A number of pharmaceutical additives did not effect the determination with RSD within ±3.1 %.     

Simultaneous Determination of Clobetasol Propionate and Calcipotriol in a Novel Fixed Dose Emulgel Formulation by LC-UV

A new, rapid, selective, cheap and simple RP-LC method has been developed and validated for the simultaneous determination of clobetasol propionate and calcipotriol mixtures in bulk drugs (raw materials) and in a novel-fixed dose emulgel formulation. Separation was carried out using a NovaPak C18 column with methanol:water (74:26 v/v) as mobile phase for isocratic elution at a flow rate of 1.0 mL min^?1. The column temperature was set at 25 °C. Calibration curves were established ranging between 0.5 and 20 μg mL^?1 and 0.5 and 10 μg mL^?1 for clobetasol propionate and calcipotriol, respectively. Limit of detection and limit of quantification values of the method was found as 0.16 and 0.48 μg mL^?1 for clobetasol propionate and 0.10 and 0.30 μg mL^?1 for calcipotriol, respectively. The method was validated in accordance with ICH guidelines and obtained results proved that the proposed method was precise, accurate, selective and sensitive for the simultaneous analysis of clobetasol propionate and calcipotriol. The proposed method can be easily applied for the simultaneous determination of clobetasol propionate and calcipotriol in prepared emulgel formulations. The obtained validation results showed that the RP-LC method is suitable for routine quantification of clobetasol propionate and calcipotriol in emulgel formulations with high precision and accuracy.     

Determination of Nateglinide in Human Plasma by LC-ESI-MS and Its Application to Bioequivalence Study

To evaluate the bioequivalence of nateglinide, a rapid and specific liquid chromatographic-electrospray ionization mass spectrometric method was developed and validated to determine nateglinide for human plasma samples. The analyte was detected using electrospray positive ionization mass spectrometry in the selected ion monitoring mode. Tinidazole was used as the internal standard. A good linear relationship obtained in the concentration ranged from 0.05 to 16 μg mL^?1 (r ² = 0.9993). Lower limit of quantification was 0.05 μg mL^?1 using 100 μL of plasma sample. Intra- and inter-day relative standard deviations were 2.1–7.5 and 4.7–8.9%, respectively. Among the pharmacokinetic data obtained, T _max was 2.09 ± 1.06 h for reference formulation and 2.40 ± 0.97 h for test formulation. C _max was 4.17 ± 1.31 μg mL^?1 for reference formulation and 4.37 ± 1.53 μg mL^?1 for test formulation. The half-life (t _½) was 1.93 ± 0.44 h for reference formulation and 1.92 ± 0.29 h for test formulation. AUC_0–10h was 13.67 ± 4.36 μg h mL^?1 for reference formulation and 13.21 ± 4.09 μg h mL^?1 for test formulation. This method was successfully applied to the pharmacokinetic study in human plasma samples.     

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.     

Simultaneous Determination of Benzodiazepines and Ketamine from Alcoholic and Nonalcoholic Beverages by GC-MS in Drug Facilitated Crimes

A GC-MS method with HP-5MS capillary column was developed for the simultaneous determination of underivatized flunitrazepam, clonazepam, alprazolam, diazepam and ketamine from drinks by extraction with chloroform: isopropanol 1:1 (v/v). All linearity ranges were between 50 and 1,000 μg mL^?1 for all compounds both in beer and in peach juice. Limit of detection was between 1.3 and 34.2 μg mL^?1, limit of quantification was between 3.9 and 103.8 μg mL^?1, the range of recoveries was 73.0 and 112.6% for all drugs in both beverages. The reported method was sensitive, rapid, and suitable for the analysis of the spiked drinks as evidence of sexual assault and robbery phenomena.     

Quantitative Determination of Phillyrin in Human Plasma Using HPLC with Fluorescence Detection

A simple, rapid, and selective high-performance liquid chromatography method for determination of phillyrin in human plasma was developed. After extracting from the plasma samples with ethyl acetate, the analyte was chromatographed on a C18 column with methanol–water (50:50, v/v, pH 2.86) as mobile phase. The fluorescence excitation and emission wavelengths were 277 and 315 nm, respectively. The linear range of the standard curve of phillyrin was 0.0313–8.0 μg mL^?1 (r > 0.999). The limit of detection was 6.31 ng mL^?1. The average recovery of phillyrin was 101.02% from plasma. The intra- and inter-day variabilities of phillyrin were <10.00%.     

LC Method with Precolumn Derivatization for Analysis of Mexiletine in Pharmaceutical Preparations

A isocratic, selective and accurate LC method of analysis of mexiletine in pharmaceutical preparations has been developed and validated. The method is based on derivatization of mexiletine with 4-chloro-7-nitrobenzofurazan in pH 9.0 borate buffer to yield a yellow product. Chromatography was performed on a C₁₈ column (150 × 4.6 mm i.d.) with acetonitrile–water 80:20 (v/v) as mobile phase at a flow rate of 1.0 mL min^?1. UV–visible absorbance detection was performed at 458 nm. The retention time of the mexiletine derivative was 4.10 min, and response was a linear function of concentration in the range 0.5–4.0 μg mL^?1 (r = 0.9998). The limits of detection and quantification were 0.05 and 0.15 μg mL^?1, respectively. Method validation revealed precision, sensitivity, and robustness were acceptable. Low RSD values are indicative of high precision, and high recovery values are indicative of the accuracy of the method. Results obtained by use of the proposed method for analysis of the mexiletine content of pharmaceutical a preparation were compared with those obtained by use of the official method. The method has been used for analysis of pharmaceutical preparations.     

Simultaneous Determination of Five Major Compounds in Polygonum cuspidatum by HPLC

An RP-HPLC method was developed for the first time to simultaneously determine five major compounds in Polygonum cuspidatum, namely resveratrol, polydatin, anthraglycoside B, emodin and physcion with UV detection at 306 nm. The column was an Agilent Zorbax SB-C₁₈ (250 × 4.6 mm i.d., 5 μm). The separation was carried out with a gradient program. The mobile phase was acetonitrile–water (containing 0.1% formic acid) at a flow rate of 1.0 mL min^?1. The standard curve was rectilinear in the range of 2.04–62.96 μg mL^?1 (r = 0.9998) for resveratrol, 20.13–239.7 μg mL^?1 (r = 0.9998) for polydatin, 7.19–71.92 μg mL^?1 (r = 1.0000) for anthraglycoside B, 2.68–83.68 μg mL^?1 (r = 0.9998) for emodin and 0.60–14.37 μg mL^?1 (r = 0.9997) for physcion. The recoveries of the markers were 96.0, 106.5, 97.8, 97.9 and 98.1%, respectively. The relative standard deviation of intra-day and inter-day were less than 5.0 and 2.3%. This method was simple, accurate and reproducible. The developed method was successfully applied to analyze five compounds in P. cuspidatum of 20 commercial brands.     

LC Determination of a Novel Synthetic Thiazolidinedione (BP-1107) in Rat Plasma and Its Application to a Pharmacokinetic Study

A simple, rapid, sensitive and reliable liquid chromatographic method for the quantification of BP-1107 in rat plasma has been established. Plasma samples were prepared by extraction with tert-butyl methyl ether, and troglitazone was used as an internal standard. The analytical separation was performed on a C₁₈ column using acetonitrile–0.3% phosphoric acid in water (pH 4.00 adjusted with triethylamine) (75:25, v/v) as a mobile phase. A detailed validation of the method was performed as per USFDA guidelines. For BP-1107 at the concentrations of 2.42, 16.11 and 32.22 μg mL^?1 in rat plasma, the extraction recoveries were 114.14 ± 9.75, 95.37 ± 12.06 and 90.00 ± 6.46%, respectively. The mean recovery for internal standard was 91.96 ± 2.51%. The lower limit of quantitation of BP-1107 was 16 ng. The linear quantification range of the method was 0.81–53.70 μg mL^?1 in rat plasma with a correlation coefficient greater than 0.999. The intra-day and inter-day accuracy for BP-1107 at 2.42, 16.11 and 32.22 μg mL^?1 levels in rat plasma fell between 97.10–110.02 and 97.52–108.04%. The intra-day and inter-day precision were in the ranges of 1.91–5.63 and 4.43–6.28%, respectively. The method was successfully applied to a pharmacokinetic study of BP-1107 in rats after an intravenous administration.     

UPLC-PDA Analysis for Simultaneous Quantification of Four Active Compounds in Crude and Processed Rhizome of Polygonum multiflorum Thunb

A novel, rapid and specific ultra performance liquid chromatography-photo diode array detection method was developed for the simultaneous determination of 2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside (TSG), emodin-8-O-β-d-glucoside (EMG), emodin (EM) and physcion (PS). The chromatographic separation was performed on an Acquity BEH C₁₈ column (100 × 2.1 mm i.d., 1.7 μm). The mobile phase was a mixture of 0.3% acetic acid–water and 0.3% acetic acid–acetonitrile employing gradient elution at the flow rate of 0.4 mL min^?1. The four compounds behaved linearly in the concentration range between 60.80–3040.00 μg mL^?1 (TSG), 0.50–25.00 μg mL^?1 (EMG), 2.16–108.00 μg mL^?1 (EM) and 1.56–78.00 μg mL^?1 (PS), respectively with correlation coefficients >0.999. The precision of the method were below 5% RSD. Recoveries of the four compounds ranged from 95.71 to 102.97%, with RSD values less than 2%.