Determination of Sulfonamides in Pharmaceuticals and Rabbit Plasma by Microchip Electrophoresis with LED-IF Detection

In this paper, we describe a compact and low-cost light-emitting diode-induced fluorescence (LED-IF) detection coupled to microchip electrophoresis for the determination of sulfonamides in pharmaceutical formulations and rabbit plasma. Three fluorescein isothiocyanate-labeled sulfonamides in rabbit plasma were separated in the running buffer of 40 mM phosphate buffer (pH 7.0) at the separation voltage of 2.0 kV, and detected by LED-IF detector in which the high-power blue LED was driven at the constant current of 150 mA and the emitted fluorescence over 510 nm was collected by a planar photodiode. The linear concentration ranged from 2.0 to 125.0 μg mL^?1, both for sulfadiazine and sulfamethazine with the correlation coefficients (r ²) of 0.995 and 0.997, respectively, and from 2.0 to 100.0 μg mL^?1 with the correlation coefficients (r ²) of 0.997 for sulfaguanidine. The limits of detection for the three sulfonamides were 0.36–0.50 μg mL^?1 (S/N = 3). Intra-day and inter-day precision of migration time and peak area for the determination of sulfonamides were <4.5 %. This method has been successfully applied to the analysis of sulfonamides in pharmaceuticals, and could be used to study the pharmacokinetics of sulfonamides in rabbit.     

Development and Validation of a Stability-Indicating HPLC Method for Determination of Ciprofloxacin Hydrochloride and its Related Compounds in Film-Coated Tablets

The objective of the current study was the development and subsequent validation of a simple, sensitive, precise and stability-indicating reversed-phase HPLC method for the determination of ciprofloxacin HCl in pharmaceutical dosage forms in the presence of its potential impurities. The chromatographic separation of ciprofloxacin HCl and its related compounds was achieved on an Inertsil ODS3 column using UV detection. The optimized mobile phase consisted of phosphoric acid solution: acetonitril. The proposed method provided linear responses within the concentration range 250–750 μg mL⁻¹ for ciprofloxacin HCl and 0.5–1.5 μg mL⁻¹ for its related compounds. LOD and LOQ values for the active substance were 5.159 and 15.632 μg mL⁻¹, respectively. Correlation coefficients (r) of the regression equations for the impurities were greater than 0.99 in all cases. The precision of the method was demonstrated using intra- and inter-day assay RSD% values which were less than 1% in all instances. No interference from any components of pharmaceutical dosage forms or degradation products was observed.

     

LC Method for Quantification of ent-11α-Hydroxy-15-oxo-kaur-16-en-19-oic Acid in Rabbit Plasma: Validation and Application to a Pharmacokinetic Study

ent-11α-Hydroxy-15-oxo-kaur-16-en-19-oic acid (5F), a diterpenoid isolated from the Chinese herb Pteris semipinnata L, has been suggested to show antitumor properties. A simple and sensitive LC method was developed for the determination of 5F in rabbit plasma. The method involved liquid–liquid extraction using ethyl acetate under acidic conditions using naproxen as an internal standard. Separations were performed on a reversed-phase column with a mixture of 1% (v/v) glacial acetic acid and methanol (45:55, v/v) as mobile phase and UV detection was utilized at 242 nm. The calibration plot was linear in the range 0.20–10.0 μg mL⁻¹ (correlation coefficients r ² > 0.998). The detection limit was 0.20 μg mL⁻¹, mean extraction recovery was above 82%, intra-day precision of the method was less than 6.4%, and inter-day precision was better than 8.7%, respectively. The validated assay was found to be suitable for the pharmacokinetic study of 5F in rabbits.

     

Simultaneous Determination of Procaspase Activating Compound 1 and Permeability Markers in Intestinal Perfusion Samples and Application to a Rat Intestinal Absorption Study

A sensitive and simple HPLC method for simultaneous determination of PAC-1 (first procaspase-activating compound), phenol red, and permeability markers (carbamazepine and furosemide) in perfusion samples was developed and validated to assess intestinal absorption of PAC-1 using single-pass intestinal perfusion technique (SPIP) in rats. The chromatographic separation was carried out on a Kromasil C₁₈ column (150 mm × 4.6 mm, 5 μm) with acetonitrile–methanol–30 mmol L⁻¹ phosphate buffer (pH 3.0, 25:10:65, v/v/v) as mobile phase at a flow rate of 1.0 mL min⁻¹, and the wavelength of the UV detector was set at 281 nm. The calibration curves were linear in the ranges of 2.40–48.0 μg mL⁻¹ for PAC-1; 3.60–72.0 μg mL⁻¹ for carbamazepine; 3.20–64.0 μg mL⁻¹ for furosemide, and 4.80–96.0 μg mL⁻¹ for phenol red (r > 0.999). Both the intra- and inter-day precisions (RSD%) of all analytes were less than 6.8 % at three concentration levels, while accuracy ranged from 95.4 to 104.5 %. Data obtained in all method validation studies indicated that the method was suitable for the intended purpose. The effective permeability values (P _eff) considering water flux with the help of non-permeable marker phenol red was calculated to be 0.42 × 10⁻⁴, 0.62 × 10⁻⁴, 0.32 × 10⁻⁴ cm s⁻¹ for PAC-1; 0.72 × 10⁻⁴, 0.77 × 10⁻⁴, 0.52 × 10⁻⁴ cm s⁻¹ for carbamazepine; 0.20 × 10⁻⁴, 0.16 × 10⁻⁴, 0.12 × 10⁻⁴ cm s⁻¹ for furosemide in duodenum, jejunum and ileum, respectively. The P _eff value can be increased by co-perfusion with verapamil, indicating that absorption of PAC-1 is efficiently transported by P-glycoprotein (P-gp) in the gut wall.

     

Validated LC Method for Simultaneous Analysis of Tramadol Hydrochloride and Aceclofenac in a Commercial Tablet

This paper describes development and validation of a high-performance liquid chromatographic method for simultaneous analysis of tramadol hydrochloride (TR) and aceclofenac (AC) in a tablet formulation. When the combination formulation was subjected to ICH-recommended stress conditions, adequate separation of TR, AC, and the degradation products formed was achieved on a C₁₈ column with 65:35 (v/v) 0.01 M ammonium acetate buffer, pH 6.5—acetonitrile as mobile phase at a flow rate of 1 mL min⁻¹. UV detection was performed at 270 nm. The method was validated for specificity, linearity, LOD and LOQ, precision, accuracy, and robustness. The method was specific against placebo interference and also during forced degradation. The linearity of the method was investigated in the concentration ranges 15–60 μg mL⁻¹ (r = 0.9999) for TR and 40–160 μg mL⁻¹ (r = 0.9999) for AC. Accuracy was between 98.87 and 99.32% for TR and between 98.81 and 99.49% for AC. Because degradation products were well separated from the parent compounds, the method was stability-indicating.

     

Development and Validation of a Stability-Indicating LC Method for the Determination of Rupatadine in Pharmaceutical Formulations

A reversed-phase liquid chromatography (RP-LC) method was validated for the determination of rupatadine in pharmaceutical dosage forms. The LC method was carried out on a Gemini C₁₈ column (150 mm × 4.6 mm I.D.), maintained at 30 °C. The mobile phase consisted of ammonium acetate buffer (pH 3.0; 0.01 M) with 0.05% of 1-heptanesulfonic acid–acetonitrile (71.5:28.5, v/v), run at a flow rate of 1.0 mL min⁻¹ and using photodiode array (PDA) detection at 242 nm. The chromatographic separation was obtained with retention time of 5.15 min, and was linear in the range of 0.5–400 μg mL⁻¹ (r ² = 0.9999). The specificity and stability-indicating capability of the method was proven through the degradation studies and showing also, that there was no interference of the excipients. The accuracy was 100.39% with bias lower than 0.58%. The limits of detection and quantitation were 0.01 and 0.5 μg mL⁻¹, respectively. Moreover, method validation demonstrated acceptable results for precision, sensitivity and robustness. The proposed method was applied for the analysis of pharmaceutical dosage forms assuring the therapeutic efficacy.

     

Determination of Tectorigenin in Rat Plasma: Application to a Pharmacokinetic Study After Oral Administration of Tectorigenin or Its Prodrug Tectoridin

A simple, sensitive, and validated liquid chromatographic method has been developed for the determination of tectorigenin in rat plasma and application to a pharmacokinetic study after oral administration of tectorigenin or its prodrug tectoridin. The analysis was performed on a Kromasil C₁₈ analytical column using gradient elution with acetonitrile 0.1% phosphonic acid water at 0.8 mL min⁻¹. The detection wavelength for UV detection was set at 264 nm. The established method was fully validated with parameters as follows: the intra- and inter-day assay precisions (CV) of three analytes were in the range of 4.2–13.3% and accuracies were between 98.0 and 107.5%; the calibration curve was linear with r ² > 0.99 over a concentration range of 0.02–2 μg mL⁻¹; the lower limit of quantification was 0.02 μg mL⁻¹; tectorigenin showed stable in rat plasma after 12 h incubation at room temperature, 15 days storage at −80 °C and three freeze/thaw cycles, as well as in reconstitute buffer for 24 h at 25 °C; and the mean recoveries of tectorigenin were 92.3 ± 3.2, 95.5 ± 2.9 and 94.5 ± 3.0% with quality control levels of 0.02, 0.2 and 2 μg mL⁻¹, respectively. In conclusion, this method is simple, economic, and sensitive enough for in vivo pharmacokinetic studies of tectorigenin.

     

LC Evaluation of In Vitro Release of AZT from Microemulsions

A reversed phase LC method was developed and validated to analyze the in vitro release of AZT from microemulsions. A mobile phase of acetonitrile:water (15:85) was used. The method validation showed good selectivity and linearity (r = 0.9993) for sample concentrations ranging from 0.6 to 100.0 μg mL⁻¹. The RSD values (0.7–4.3%) and percentage recovery (88.1–109.8%) were within acceptable limits. The limit of detection (LOD) and limit of quantitation (LOQ) were found to be 0.012 and 0.041 μg mL⁻¹. Quantitative analysis of the values obtained in the drug release assay indicates that the microemulsions used promote sustained release of AZT, which follows a Fickian diffusion mechanism.

     

Application of Multicriteria Methodology in the Development of Improved RP-LC-DAD for Determination of Rizatriptan and Its Degradation Products

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-DAD Determination of Fleroxacin in Bulk and Pharmaceutical Dosage Forms

Fleroxacin is a third generation fluoroquinolone with broad spectrum antibacterial activity. In this work an LC-DAD method for the analysis of fleroxacin was developed and validated using UV detection at 286 nm. The method was validated for linearity, precision, robustness, LOD, LOQ, specificity and accuracy at concentrations of 0.2–20.0 μg mL⁻¹ and r ² = 1. The LOD and LOQ were 0.059 and 0.197 μg, respectively, the recoveries were 99.92–102.0% and the CV was less than 2.0%. The LC-DAD validated method provided analytical sensitivity, specificity and reproducibility suitable for quality control analysis.

     

Quantitative Analysis of Toosendanin in the Fruit of Melia toosendan Sieb. Et Zucc (Meliaceae) by High-Performance Liquid Chromatography Coupled with Charged Aerosol Detection

In this work, a simple and rapid high-performance liquid chromatography coupled with charged aerosol detector (HPLC-CAD) method was first developed for the quantitation of toosendanin, the major constituent of the dried fruit of Melia toosendan Sieb. Et Zucc. Samples were well separated on an Agilent ZOBAX SB C18 column (4.6 mm × 250 mm, 5 μm) by isocratic elution using 33 % acetonitrile and 67 % water containing 0.1 % formic acid (v/v) at the flow rate of 1.0 mL min⁻¹. The nitrogen inlet pressure of the charged aerosol detector (CAD) was 35 psi, and the nebulizer chamber temperature was 35 °C. The established method was well validated. Satisfactory linearity was achieved (r ² > 0.9997) in a relatively wide concentration range (5–500 μg mL⁻¹). The intra- and inter-day precisions, repeatability, and stability of the method were good with relative standard deviations (RSDs) of 1.05, 2.23, 2.39, and 2.03 %, respectively. The method also showed excellent accuracy with recovery rates of 97.42–101.87 %. Particularly, CAD showed much better sensitivity (LOQ 4 μg mL⁻¹) than evaporative light scattering detector (LOQ 100 μg mL⁻¹) for toosendanin’s determination. The established method was further applied in the quantitation of toosendanin in 39 batches of raw and stir-fried toosendan fructus. The HPLC-CAD method was rapid and accurate, and could be used for the routine analysis and quality control of toosendan fructus and its preparations.

     

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.

     

HPLC and UPLC Methods for the Simultaneous Determination of Enalapril and Hydrochlorothiazide in Pharmaceutical Dosage Forms

High efficiency and less elution are the basic requirements of high-speed chromatographic separation. In this study, a new gradient reverse phase chromatographic methods were developed using HPLC and UPLC systems for simultaneous determination of enalapril maleate (ENL) and hydrochlorothiazide (HCZ) in pharmaceutical dosage forms. The chromatographic separations of ENL and HCZ were achieved on a Waters μ-Bondapak C 18, (300 × 3.9 mm, 10 μm) and Waters Acquity BEH C18 (100 × 2.1 mm, 1.7 μm) columns for HPLC within 5.30 min and UPLC within a short retention time of 1.95 min, respectively. A linear response was observed over the concentration range 0.270–399 μg mL⁻¹ of ENL, 0.260–399 μg mL⁻¹ of HCZ for HPLC system and 0.270–399 μg mL⁻¹ of ENL and 0.065–249 μg mL⁻¹ of HCZ for UPLC system. Also, limit of detection for ENL was 1.848 ng mL⁻¹ and 31.477 ng mL⁻¹ for HCZ, 2.804 ng mL⁻¹ for ENL and 2.943 ng mL⁻¹ for HCZ using HPLC and UPLC, respectively. The proposed methods were validated according to ICH guideline with respect to precision, accuracy, and linearity. Forced degradation studies were also performed for both compounds in bulk drug samples to demonstrate the specificity and stability indicating power of the HPLC method. Comparison of system performance with conventional HPLC was made with respect to analysis time, efficiency, and resolution.

     

Determination of Repertaxin Enantiomeric Purity by HPLC on Chiral Stationary Phase

A precise and sensitive LC method for the determination of repertaxin enantiomeric purity has been developed and validated. Baseline separation with a resolution higher than 2.0 was accomplished within 20 min using a Chiralpak AD-H column (250 × 4.6 mm; particle size 5 μm) and n-hexane:2-propanol (90:10 v/v) as mobile phase at a flow rate of 1 mL min⁻¹. Eluted analytes were monitored by UV detection at 260 nm. The effects of mobile phase composition, temperature and flow rate on enantiomeric selectivity and on resolution of enantiomers were investigated. Calibration curves were plotted within the concentration range between 0.002 and 1.0 mg mL⁻¹ (n = 3), and relative standard deviation (RSD) of the inter-batch assay and intra-batch assay was less than 1.27 and 1.16 %. LOD and LOQ for repertaxin were 0.65 and 2.19 μg mL⁻¹; those for its enantiomer were 0.70 and 2.34 μg mL⁻¹, respectively. The method was evaluated and validated by analysis of bulk samples of repertaxin of different enantiomeric purity. It was demonstrated that the method was accurate, robust, and sensitive, and enabled practical analysis of real samples.

     

Comparison of HPLC Versus HPTLC-Densitometry for the Quantification of Chromone Glucosides in Saposhnikoviae divaricatae Radix

A HPLC and a HPTLC-densitometric method were developed for the quantification of prim-O-glucosylcimifugin and 4′-O-β-d-glucosyl-5-O-methylvisamminol the major chromone glucosides in the roots of Saposhnikovia divaricata. The validation of both methods resulted in comparable parameters regarding stability, specificity, linearity, robustness, precision and recovery, whereas complementary advantages were obtained concerning LOD and LOQ. The HPTLC-based densitometry revealed a lower LOD (1.11 versus 4.37 μg mL⁻¹ in HPLC) and LOQ (3.36 versus 13.24 μg mL⁻¹ in HPLC) for prim-O-glucosylcimifugin, whereas the HPLC resulted in a lower LOD (1.00 versus 4.10 μg mL⁻¹ in HPTLC-densitometry) and LOQ (3.04 versus 12.46 μg mL⁻¹ in HPTLC-densitometry) for 4′-O-β-d-glucosyl-5-O-methylvisamminol. Both methods revealed nearly matching contents of the chromones after analysis of different commercially available batches of Saposhnikoviae divaricatae radix with a total content for both chromone glycosides in the range from 0.31 ± 0.011 to 0.56 ± 0.021 % determined by HPLC and between 0.34 ± 0.011 and 0.61 ± 0.009 % determined by HPTLC. The plant material cultivated in Germany showed a very similar content and ratio of both chromone glucosides in comparison to the standard batches originating from China.

     

Pharmacokinetics and Tissue Distribution Study of Tryptanthrin in Rats by RP-HPLC with DAD Detector

A simple RP-LC-UV method was established for the determination of tryptanthrin in plasma and different tissues of rats. The separation was achieved by HPLC on a C₁₈ column with a mobile phases composed of acetonitrile–water (47:53, v/v), UV detection was used at 251 nm. Good linearity was found between 0.0183–1.1712 μg mL⁻¹ (r ² = 0.999) for plasma and 0.0937–1.7568 μg mL⁻¹ for the tissue samples, respectively (r ² ≥ 0.9932). The intra- and inter-day precisions expressed as the relative standard deviation for the method were 0.92–6.01 and 1.06–9.11 %, respectively. The relative recoveries of tryptanthrin ranged from 95.26 to 97.89 % for plasma and 82.55 to 114.99 % for tissue homogenates (except heart). The developed method was successfully applied to the pharmacokinetics and tissue distribution research after orally administration of a 56-mg kg⁻¹ dose of tryptanthrin to healthy SD rats. The main pharmacokinetics distribution results showed that liver, lung, small intestine, and large intestine were the major distribution tissues of tryptanthrin in rats, and that tryptanthrin had difficulty in crossing the blood–brain barrier.

     

Simultaneous Determination of Antibiotics in Anti-Acne Cosmetics by Rapid LC with DAD

A suitable method that allows, for the first time, the simultaneous determination of nine antibiotics which may help the therapy of acne vulgaris by rapid liquid chromatography with diode array detection in 7 min is presented in this work. An SB RP18 (50 × 4.6 mm; 1.8 μm particle size) column was used with the mobile phase consisting of a mixture of 0.1 mol L⁻¹ potassium dihydrogen phosphate (pH 2.5) and acetonitrile at the gradient elution program. The correlation coefficients were all above 0.9999 in the linear range between 4–100 μg mL⁻¹, the average spiked recoveries (n = 6) were 92.2–103.2% with RSD ranging from 0.04 to 4.5% depending on the target analytes. The method detection limits were in the range of 0.02–0.2 μg mL⁻¹ in anti-acne cosmetics. The analysis of real cosmetic preparations demonstrated the fitness for the whole analytical procedure. The proposed method appeared therefore as a sound alternative for official testing method, which could overcome the general problems of time consuming, lack of the specificity and precision difficulty.

     

RP-LC-UV Determination of Proanthocyanidins in Guazuma ulmifolia

A simple, reproducible, and efficient liquid chromatographic method was developed with UV detection. Water (0.05% TFA):acetonitrile (0.05% TFA) was used as the mobile phase in a gradient system for the determination of procyanidin B2 (PB2) and epicatechin (EC) in the bark of Guazuma ulmifolia Lam. The analysis was performed using a Phenomenex Gemini RP C₁₈ column (5 μm) as stationary phase, at 30 °C, with a flow rate of 0.8 mL min⁻¹, at a wavelength of 210 nm for detection and determination. The main validation parameters of the method were also determined. Calibration curves were found to be linear, with ranges of 20.00–150.00 (PB2) and 10.00–110.00 μg mL⁻¹ (EC). The correlation coefficients of linear regression analysis were between 0.9981 and 0.9988, and the detection limits were between 2.89 and 2.54 μg mL⁻¹. The contents of PB2 and EC were successfully determined, with satisfactory reproducibility and recovery. Recoveries of the PB2 and EC were 103.00 and 104.01%, respectively. The method was successfully applied to the determination of procyanidins in the bark of G. ulmifolia.

     

Quantitative Determination of Busulfan in Human Plasma by UPLC

A rapid and reliable UPLC method was developed and validated for the determination of busulfan in human plasma. After protein precipitation, derivatization, and liquid–liquid extraction, separation of derivatized busulfan was achieved on an Acquity BEH C₁₈ column using a gradient mobile phase consisting of a trifluoroacetic acid aqueous solution (0.2%, v/v) and acetonitrile. The column temperature was maintained at 50 °C and UV detection was carried out at 254 nm. The complete analytical run time was 1.3 min, 7-fold faster than our previous LC methodology. Quantification was performed using external standardization and calibration curves were linear (r ≥ 0.999) over the dynamic range of 0.05–5.00 μg mL⁻¹. Intra-day and inter-day coefficients of variation were ≤6.9 and 3.9%, respectively, across the range of concentrations. Accuracy of the analytical method expressed as the relative error percentage was better than 5.4%. LOD and LOQ were 0.013 and 0.025 μg mL⁻¹, respectively. Data obtained using the UPLC method was compared to those obtained from our previously used LC method by Deming regression analysis. The UPLC method was accurate, sensitive, and greatly increased sample analysis throughput as compared to our previous LC methodology allowing for a 4-fold increase in the number of patients who could be monitored during transplant therapy.