Randa A. Abdel Salam

Rapid and simultaneous determination of antioxidant markers and caffeine in commercial teas and dietary supplements by HPLC-DAD.

A simple and fast reverse-phase high-performance liquid chromatography procedure coupled with photodiode array detector (RP-HPLC-DAD) was developed and validated for the analysis of major catechins, proanthocyanidin (procyanidin B2) and caffeine in 25 different natural complex matrices containing Camellia sinensis L. and/or grape seed extracts, two popular plant extracts that have been widely used as natural antioxidants in various food and beverage applications. Using an isocratic elution system, separation of all compounds was achieved within 12 min. Excellent linearity was observed for all of the standard calibration curves, and the correlation coefficients were above 0.9997. Limits of detection for all of the analyzed compounds ranged between 2.80×10(-3) and 2.51×10(-2) μg mL(-1); limits of quantitation ranged between 9.30×10(-3) and 8.36×10(-2) μg mL(-1). The developed method was found to be accurate and sensitive and is ideally suited for rapid, routine analysis of principal components in these well-known natural antioxidants.

Validated and Optimized High-Performance Liquid Chromatographic Determination of Tizoxanide, the Main Active Metabolite of Nitazoxanide in Human Urine, Plasma and Breast Milk

A high-performance liquid chromatographic method was optimized and validated for the determination of desacetyl nitazoxanide (tizoxanide), the main active metabolite of nitazoxanide in human plasma, urine and breast milk. The proposed method used a CN column with mobile phase consisting of acetonitrile-12mM ammonium acetate-diethylamine in the ratio of 30:70:0.1 (v/v/v) and buffered at pH 4.0 with acetic acid, with a flow rate of 1.5 mL/min. Quantitation was achieved with UV detection at 260 nm using nifuroxazide as internal standard. A simplified direct injection of urine samples without extraction in addition to the urinary excretion pattern were calculated using the proposed method. Also, the effectiveness of protein precipitation and a clean-up procedure were investigated for biological plasma and human breast milk samples. The validation study of the proposed method was successfully carried out in an assay range between 0.2 and 20 µg/mL.

Chemometric-assisted spectrophotometric methods and high performance liquid chromatography for simultaneous determination of seven β-blockers in their pharmaceutical products: a comparative study.

Chemometric-assisted spectrophotometric methods and high performance liquid chromatography (HPLC) were developed for the simultaneous determination of the seven most commonly prescribed β-blockers (atenolol, sotalol, metoprolol, bisoprolol, propranolol, carvedilol and nebivolol). Principal component regression PCR, partial least square PLS and PLS with previous wavelength selection by genetic algorithm (GA-PLS) were used for chemometric analysis of spectral data of these drugs. The compositions of the mixtures used in the calibration set were varied to cover the linearity ranges 0.7-10 μg ml(-1) for AT, 1-15 μg ml(-1) for ST, 1-15 μg ml(-1) for MT, 0.3-5 μg ml(-1) for BS, 0.1-3 μg ml(-1) for PR, 0.1-3 μg ml(-1) for CV and 0.7-5 μg ml(-1) for NB. The analytical performances of these chemometric methods were characterized by relative prediction errors and were compared with each other. GA-PLS showed superiority over the other applied multivariate methods due to the wavelength selection. A new gradient HPLC method had been developed using statistical experimental design. Optimum conditions of separation were determined with the aid of central composite design. The developed HPLC method was found to be linear in the range of 0.2-20 μg ml(-1) for AT, 0.2-20 μg ml(-1) for ST, 0.1-15 μg ml(-1) for MT, 0.1-15 μg ml(-1) for BS, 0.1-13 μg ml(-1) for PR, 0.1-13 μg ml(-1) for CV and 0.4-20 μg ml(-1) for NB. No significant difference between the results of the proposed GA-PLS and HPLC methods with respect to accuracy and precision. The proposed analytical methods did not show any interference of the excipients when applied to pharmaceutical products.

Determination of Acid and Neutral Cannabinoids in Extracts of Different Strains of Cannabis sativa Using GC-FID

Cannabis (Cannabis sativa L.) is an annual herbaceous plant that belongs to the family Cannabaceae. Trans-Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) are the two major phytocannabinoids accounting for over 40% of the cannabis plant extracts, depending on the variety. At the University of Mississippi, different strains of C. sativa, with different concentration ratios of CBD and Δ9-THC, have been tissue cultured via micropropagation and cultivated. A GC-FID method has been developed and validated for the qualitative and quantitative analysis of acid and neutral cannabinoids in C. sativa extracts. The method involves trimethyl silyl derivatization of the extracts. These cannabinoids include tetrahydrocannabivarian, CBD, cannabichromene, trans-Δ8-tetrahydrocannabinol, Δ9-THC, cannabigerol, cannabinol, cannabidiolic acid, cannabigerolic acid, and Δ9-tetrahydrocannabinolic acid-A. The concentration-response relationship of the method indicated a linear relationship between the concentration and peak area ratio with R2 > 0.999 for all 10 cannabinoids. The precision and accuracy of the method were found to be ≤ 15% and ± 5%, respectively. The limit of detection range was 0.11 - 0.19 µg/mL, and the limit of quantitation was 0.34 - 0.56 µg/mL for all 10 cannabinoids. The developed method is simple, sensitive, reproducible, and suitable for the detection and quantitation of acidic and neutral cannabinoids in different extracts of cannabis varieties. The method was applied to the analysis of these cannabinoids in different parts of the micropropagated cannabis plants (buds, leaves, roots, and stems).

HPLC–DAD Determination of Seven Antioxidants and Caffeine in Different Phytopharmaceuticals

A high-performance liquid chromatography method employing diode array detection was developed to determine levels of the major catechins, proanthocyanidin (procyanidin B2), caffeine, thymoquinone and carvacrol and its isomer, thymol, which are present in different natural complex matrices found in commercial products of Camellia sinensis L. and/or Nigella sativa L. Reversed-phase separation was performed on a C18 column by using gradient elution by varying the proportions of solvent A (distilled water containing 0.05% orthophosphoric acid) and solvent B (acetonitrile), with a flow rate of 1.5 mL/min and duration of 31 min. Excellent linearity was observed for all standard calibration curves, and correlation coefficients were above 0.9996. The developed method is efficient, with high reproducibility and sensitivity, and is ideally suited for rapid and routine analysis of principal components in these promising medicinal plants.

QUANTITATIVE DETERMINATION OF LEVETIRACETAM IN HUMAN URINE USING HPLC-UV AND ITS IDENTIFICATION BY LC-ESI-MS

A rapid method for the quantification of levetiracetam (LV) in human urine using high-performance liquid chromatography combined with UV detection (HPLC–UV) was developed and validated for linearity, repeatability, limit of detection, and limit of quantitation. In addition, a high-performance liquid chromatography combined with electrospray ionization mass spectrometry was described for its identification in human urine. Chromatographic separation was achieved on 250 × 4.6 mm (i.d.) Discovery® (5 µm particle size) reversed-phase C18 analytical column using 0.1% formic acid in water–acetonitrile, in ratio of 85:15, v/v as mobile phase and UV detection at 210 nm for the HPLC–UV method and using 0.1% formic acid in water–acetonitrile, in ratio of 75:25, v/v as mobile phase for LC-ESI-MS method. The HPLC–UV method was successfully applied for establishment of the urinary excretion pattern of LV after oral dose.

VALIDATED STABILITY-INDICATING HPTLC AND HPLC METHODS FOR DETERMINATION OF PIPAZETHATE AND ITS DEGRADANT

This paper describes validated high-performance thin-layer chromatography and high-performance liquid chromatography methods for the simultaneous determination of pipazethate hydrochloride (PZ) and its degradant. The HPTLC separation was carried out on Merck HPTLC aluminum sheets of silica gel 60 F254 as stationary phase using chloroform:diethylamine:methanol (9.4:0.1:0.5, v/v/v) as a mobile phase. Quantification was achieved using densitometric measurements at 225 nm. The isocratic reversed phase HPLC separation was performed on 5 µm CN column (Luna, Phenomenex®, USA.). Good resolution between PZ and its degradants was achieved using a mixture of acetonitrile:12 mM ammonium acetate:diethylamine (35:65:0.1, v/v/v, pH 4.0) as a mobile phase. Quantitation was achieved with UV detection at 225 nm based on peak area. Forced degradation studies were performed on bulk sample of PZ using acid (1N hydrochloric acid), alkaline (0.1N sodium hydroxide), oxidation (0.33% hydrogen peroxide), heat (75°C), and photolytic degradation. The proposed HPLC method was utilized to investigate the kinetics of acidic, alkaline, and oxidative degradation processes of PZ at different temperatures and the apparent pseudo-first-order rate constant, half-life, and activation energy were calculated. The pH-rate profile of degradation of PZ in Britton-Robinson buffer solutions within the pH range 1.8–11.4 was studied.

Simultaneous determination of selected veterinary antibiotics in Nile tilapia (Orechromis niloticus) and water samples by HPLC/UV and LC-MS/MS

A method was optimized and validated for simultaneous estimation of some antibiotics such as chlortetracycline (CTC), doxycycline (DOX), florfenicol (FF), flumequine (FLU), nalidixic acid (NAL), sulfadiazine (SDI), sulfathiazole (STZ) and trimethoprim (TMP) in fish muscle and water samples. The method is based on solid phase extraction (SPE) and simple extraction followed by high performance liquid chromatography with ultraviolet detection (HPLC-UV). The HPLC method was optimized using experimental design. The optimum conditions for separation determined with the aid of central composite design were: (1) initial mobile phase concentration: 0.1% formic acid in water/acetonitrile (90/10, v/v), (2) column temperature 25 °C and (3) mobile phase flow rate (1.2 ml min−1). The optimized method was validated according to ICH guidelines. The detection and quantification limits were between 0.2–0.4 and 0.3–0.6 μg kg−1, respectively, for fish and between 0.005–0.02 and 0.01–0.08 μg ml−1, respectively, for water. The procedure was also applied to the analysis of spiked Nile tilapia samples. Three antibiotics (SDI, CTC and FF) were orally administered and the residue was analyzed using liquid chromatography-electro spray ionization-mass spectrometry with positive ion mode (LC-ESI/MS).

High-performance thin-layer chromatography method for the simultaneous determination of itopride, pantoprazole, and mosapride in their formulations and spiked human plasma

The combination of itopride (ITP), pantoprazole (PAN), and mosapride (MS) is widely used in the treatment of many gastrointestinal tract (GIT) disorders. For that purpose, a new, simple, precise, accurate, and rapid high-performance thin-layer chromatography (HPTLC) method was developed and validated for the simultaneous determination of ITP, PAN, and MS in their pharmaceutical formulations. The method used Merck HPTLC aluminum plates precoated with silica gel 60 F254 as the stationary phase. The mobile phase consisted of methylene chloride—ethyl acetate—methanol—ammonia (25%) (12:2:0.8:0.2, v/v); this system was found to give compact spot of itopride (RF value of 0.22 ± 0.008), pantoprazole (RF value of 0.41 ± 0.006), and mosapride (RF value of 0.62 ± 0.029). The wavelength of thin-layer chromatography (TLC) scanner was set at 289 nm for both detection and quantitation. The calibration curves were linear over the range of 100–1500 ng spot−1 for ITP and MS, and 70–1500 ng spot−1 for PAN. The detection limits were 32.5, 16.8, and 29.8 for ITP, PAN, and MS, and the quantitation limits were 98.5, 50.3, and 90.5 for ITP, PAN, and MS. The proposed analytical method was validated according to the International Conference on Harmonization (ICH) guidelines, and the results were acceptable. The proposed method has been successfully applied for the determination of the studied drugs in their pharmaceutical preparations as well as in spiked human plasma and it gave excellent percent of recovery. The results showed excellent agreement with the reported method with respect to precision and accuracy.

Stability-indicating high-performance thin-layer chromatographic determination of ondansetron in pure form and pharmaceutical formulations

A new, simple, precise, accurate, stability-indicating, and rapid high-performance thin-layer chromatography (HPTLC) method was developed and validated for the determination of ondansetron hydrochloride in pure form and pharmaceutical formulations. The method used Merck HPTLC aluminum plates precoated with silica gel 60 F254 as the stationary phase. The mobile phase consisted of chloroform–methanol–ethyl acetate (7:2:1, v/v). This system was found to give a compact spot of ondansetron (RF value of 0.67 ± 0.011); ondansetron was subjected to acid and alkali hydrolysis, oxidation, and photodegradation. The wavelength of TLC scanner was set at 302 nm for both detection and quantitation. The calibration curves were linear over the range of 25–500 ng spot−1. The limit of detection was 4.9 ng spot−1, and the limit of quantitation was 14.7 ng spot−1. The proposed analytical method was validated according to the International Conference on Harmonization (ICH) guidelines, and the results were acceptable. The proposed method has been successfully applied to the determination of the studied drug in its pharmaceutical preparations and it gave excellent % of recovery. The results showed excellent agreement with the reported method with respect to precision and accuracy.