Validated HPLC method for quantifying permethrin in pharmaceutical formulations

An isocratic HPLC method for permethrin determination in raw material and pharmaceutical presentations as lotion and shampoo has been developed and validated following ICH recommendations. Cis and trans- isomers, impurities and degradation products are well separated. The chromatographic analysis were performed on a 4 μm particle C-18 Nova-Pak (Waters, Madrid, Spain) column (15×0.39 cm) kept in a Biorad column oven at 35°C. Mobile phase consisted of methanol–water (78:22, v/v) at a flow rate of 1 ml/min. UV detection was performed at 272 nm and peaks were identified with retention times as compared with standards and confirmed with characteristic spectra using the photodiode array detector.     

Evaluation of impurities level of perindopril tert-butylamine in tablets

Perindopril tert-butylamine is a new member of angiotensin-converting enzyme inhibitors group used in the treatment of hypertension and heart failure. In this paper, the evaluation of reversed-phase high-performance liquid chromatographic method (RP-HPLC) for the determination of impurities level of perindopril tert-butylamine in tablets was done. The chromatograms were recorded using a Hewlett Packard 1100 chromatographic system with DAD detector. Separations were performed on a YMC-Pack C8 column (250 mm × 4.6 mm; 5 μm particle size) at 50 °C column temperature. Mobile phase was a mixture of acetonitrile–potassium phosphate buffer (0.05 M) (37:63, v/v) (pH 2.5). pH of the mobile phase was adjusted with ortophosphoric acid. Mixture of acetonitrile–water (40:60, v/v) was used as a solvent. Injection volume was 50 μl, flow rate 1.7 ml min⁻¹ and UV-detection was performed at 215 nm. The developed method subjected to method validation and parameters in terms of selectivity, linearity, precision, accuracy, limit of detection, limit of quantitation and robustness were defined. The validated method is suitable for the simultaneous determination of perindopril tert-butylamine as well as its impurities in pharmaceuticals.     

Development and validation of HPLC method for determination of indomethacin and its two degradation products in topical gel

Indomethacin forms by decomposition two degradation products: 4-chlorobenzoic acid and 5-methoxy-2-methylindoleacetic acid. They have to be monitored together with an active substance both during manufacturing process and storage of pharmaceuticals. European Pharmacopoeia (Ph. Eur. 4) describes titration method for determination of indomethacin, which is not very convenient in this case for practical use. Therefore, high performance liquid chromatography is the method-of-choice enabling determination of active substance and its degradation products during one-step procedure simultaneously and automatically.

We have developed a fast, simple and fully automated analytical method for determination of indomethacin and its two impurities in pharmaceutical preparation using HPLC with UV detection. Various stationary phases were tested, especially new types of Zorbax columns made by Agilent. While the conventional C18 stationary phases were not convenient enough to achieve quick and reliable separation, Zorbax-Phenyl analytical column (75 mm × 4.6 mm, 3.5 μm) enables separation of indomethacin and its two degradation products during 7.5 min. Chromatography was performed using isocratic elution with binary mobile phase composed of acetonitrile and 0.2% phosphoric acid (50:50, v/v) at flow rate 0.6 ml/min. Even faster separation of standards was obtained with analytical column Zorbax SB-CN (150 mm × 4.6 mm, 5 μm). The separation was effected with mobile phase of the same composition, only the flow rate was increased to 1.2 ml/min. The analytical run was shortened to 5 min. Both methods use detection wavelength 237 nm and both can use either ketoprofen or flurbiprofen as internal standard for quantitation.

The first method was finally chosen for validation because of the occurrence of placebo interferences in the case of using Zorbax SB-CN. System suitability parameters and validation parameters including method precision, accuracy, linearity, selectivity and robustness were set up. Afterwards, the method was successfully applied for the practical determination of indomethacin and its degradation products in a topical gel and for compound degradation control during stability studies.     

High-performance liquid chromatographic determination of ursodeoxycholic acid after solid phase extraction of blood serum and detection-oriented derivatization

Ursodeoxycholic acid (3, 7β-dihydroxy-5β-cholanoic acid, UDCA) is a therapeutically applicable bile acid widely used for the dissolution of cholesterol-rich gallstones and in the treatment of chronic liver diseases associated with cholestasis. UDCA is more hydrophilic and less toxic than another therapeutically valuable bile acid, chenodeoxycholic acid (CDCA), the 7-epimer of UDCA. Procedures for sample preparation and HPLC determination of UDCA in blood serum were developed and validated. A higher homologue of UDCA containing an additional methylene group in the side chain was synthetized and used as an internal standard (IS). Serum samples with IS were diluted with a buffer (pH=7). The bile acids and IS were captured using solid phase extraction (C18 cartridges). The carboxylic group of the analytes was derivatized using 2-bromo-2′-acetonaphthone (a detection-oriented derivatization), and reaction mixtures were analyzed (HPLC with UV 245 nm detection; a 125–4 mm column containing Lichrospher 100 C18, 5 μm; mobile phase: acetonitrile–water, 6:4 (v/v)). Following validation, this method was used for pharmacokinetic studies of UDCA in humans.     

Determination of imperatorin in rat plasma by reversed-phase high-performance liquid chromatography after oral administration of Radix Angelicae dahuricae extract

A simple high-performance liquid chromatographic (HPLC) method has been developed for the determination of imperatorin in rat plasma and applied to a pharmacokinetic study in rats after administration of Radix Angelicae dahuricae extract. After addition of fluocinonide as an internal standard (IS), plasma samples were extracted with diethyl ether. HPLC analysis of the extracts was performed on a Diamonsil C18 analytical column using methanol–water (70:30, v/v) as the mobile phase. The UV detector was set at 254 nm. The standard curve was linear over the range 0.04–4.0 μg/mL. The lower limit of quantification was 0.04 μg/mL. The HPLC method developed could be easily applied to the determination and pharmacokinetic study of imperatorin in rat plasma after giving the animals Radix Angelicae dahuricae extract.     

Development and application of a validated HPLC method for the determination of gabapentin and its major degradation impurity in drug products

A simple isocratic reversed-phase HPLC method for the determination of gabapentin and its major degradation impurity, 3,3-pentamethylene-4-butyrolactam, was developed and validated for use in the analysis of pharmaceutical tablets and capsules. Separation was achieved on a Brownlee Spheri-5 Cyano column using an acetonitrile–10 mM KH₂PO₄/10 mM K₂HPO₄ (pH 6.2) (8:92, v/v) mobile phase. The compounds were eluted isocratically at a flow rate of 1 mL/min. Both compounds were analyzed with UV detection at 210 nm. The method was validated according to USP Category I requirements for gabapentin and USP Category II for 3,3-pentamethylene-4-butyrolactam. The validation characteristics included accuracy, precision, linearity, range, specificity, limit of quantitation and robustness. Validation acceptance criteria were met in all cases. This method was used successfully for the quality assessment of four gabapentin drug products.     

New method for high-performance liquid chromatographic determination of amantadine and its analogues in rat plasma

A novel precolumn derivatization method is described for the quantitative determination of amantadine, rimantadine and memantine in biological samples by HPLC with UV detection. The derivatization was performed at room temperature using anthraquinone-2-sulfonyl chloride (ASC) as reagent for only 10 min and without postderivatization treatment to inactivate excess reagent. The derivatives were analyzed by isocratic HPLC with a UV detector at 256 nm on a Lichrosper C18 column. The linear range for the determination of three drugs spiked in plasma (0.2 ml) was 0.05–5.0 μg/ml for amantadine and rimantadine, 0.05–2.0 μg/ml for memantine, respectively. The limits of detection and quantification were 20 and 50 ng/ml for the analytes, respectively. Application of the method to the analysis of amantadine, rimantadine and memantine in rat plasma and pharmacokinetic studies are demonstrated and proved feasible.     

Validation of a RP-HPLC method for the assay of formoterol and its related substances in formoterol fumarate dihydrate drug substance

A stability-indicating reversed-phase high performance liquid chromatographic (HPLC) method has been developed and validated for the assay of formoterol fumarate and the related substances, namely, formoterol fumarate desformyl and formoterol fumarate acetamide analogs, in the active pharmaceutical ingredient. The separation was achieved by isocratic elution using an Alltech Alltima C₁₈ (150×4.6 mm) column, a mobile phase consisting of ammonium acetate (50 mM; pH 5.0)–ethanol (65:35, v/v), a flow rate of 1.0 ml/min and UV detection at 242 nm. The detection and quantitation limits were 0.03 and 08 μg/ml, respectively, while the linear range of detection was between 0.03 and 255 μg/ml. Comparative determinations of formoterol fumarate in three lots of bulk drugs using the proposed HPLC method and the standard potentiometric titration method of pharmacopoeia show that both methods are equivalent for pure drug substance assay. However, the HPLC method allowed the separation and quantitation of the impurities not achievable with the official methods in the bulk drugs. This study shows that the proposed method is accurate, linear, and sensitive as stability indicating assay method for formoterol fumarate in the bulk drug.     

高效液相色谱法分离分析链霉素及其杂质

用离子对反相高效液相色谱法可从国内外商品链霉素中分离出包括剧毒的、致敏的及色素原部份等17种杂质,并可定量测定链霉素、链霉胍及链胍双氢链糖。由于此法系分离后再测定,故不受其他物质的干扰,较微生物法及化学比色法专属性高,且可用于测定原料、成品、发酵液及分离纯化各阶段的液体样品。     

Stability-indicating methods for the determination of piretanide in presence of the alkaline induced degradates

Stability-indicating high performance liquid chromatography (HPLC), thin-layer chromatography (TLC) and first-derivative of ratio spectra (1DD) methods are developed for the determination of piretanide in presence of its alkaline induced degradates. HPLC method depends on separation of piretanide from its degradates on μ-Bondapak C₁₈ column using methanol:water:acetic acid (70:30:1, v/v/v) as a mobile phase at flow rate 1.0 ml/min and UV detector at 275 nm. TLC densitometic method is based on the difference in Rf-values between the intact drug and its degradates on thin-layer silica gel. Iso-propanol:ammonia 33% (8:2, v/v) was used as a developing mobile phase and the chromatogram was scanned at 275 nm. The derivative of ratio spectra method (1DD) depends on the measurement of the absorbance at 288 nm in the first-derivative of ratio spectra for the determination of the cited drug in the presence of its degradates. Calibration graphs of the three suggested methods are linear in the concentration ranges 0.02–0.3 μg/20 μl, 0.5–10 μg/spot and 5–50 μg/ml, with mean percentage recovery 99.27 ± 0.52, 99,17 ± 1.01 and 99.65 ± 1.01%, respectively. The three proposed methods were successfully applied for the determination of piretanide in bulk powder, laboratory-prepared mixtures and pharmaceutical dosage form with good accuracy and precision. The results were statistically analyzed and compared with those obtained by the official method. Validation of the method was determined with favourable specificity, linearity, precision, and accuracy was assessed by applying the standard addition technique.     

High performance liquid chromatographic and thin layer densitometric methods for the determination of risperidone in the presence of its degradation products in bulk powder and in tablets

Two reproducible stability indicating methods were developed for the determination of risperidone (RISP) in presence of its degradation products in pure form and in tablets. The first method was based on reversed phase high performance liquid chromatography (HPLC), on Lichrosorb RP C 18 column (250 mm i.d., 4 mm, 10 μm), using methanol:0.05 M potassium dihydrogen phosphate pH 7 (65:35 (v/v)) as the mobile phase at a flow rate of 1 ml min⁻¹ at ambient temperature. Quantification was achieved with UV detection at 280 nm over a concentration range of 25–500 μg ml⁻¹ with mean percentage recovery of 99.87 ± 1.049. The method retained its accuracy in the presence of up to 90% of RISP degradation products. The second method was based on TLC separation of RISP from its degradation products followed by densitometric measurement of the intact drug spot at 280 nm. The separation was carried out on aluminum sheet of silica gel 60F₂₅₄ using acetonitrile:methanol:propanol:triethanolamine (8.5:1.2:0.6:0.2 (v/v/v/v)), as the mobile phase, over a concentration range of 2–10 μg per spot and mean percentage recovery of 100.1 ± 1.18. The two methods were simple, precise, sensitive and could be successfully applied for the determination of pure, laboratory prepared mixtures and tablets. The results obtained were compared with the manufacturer's method.     

Development of a HPLC method for the determination of cyclosporin-A in rat blood and plasma using naproxen as an internal standard

An isocratic reversed phase high-performance liquid chromatographic (HPLC) method with ultraviolet detection at 205 nm has been developed for the determination of cyclosporin-A (CyA) in rat blood and plasma. Naproxen was successfully used as an internal standard. Blood or plasma samples were pretreated by liquid–liquid extraction with diethyl ether. The ether extract was evaporated and the residue was reconstituted in acetonitrile–0.04 M monobasic potassium phosphate buffer (pH 2.5) solvent mixture. After washing with n-hexane, 30 μl of the reconstituted solution was injected into HPLC system. Good chromatographic separation between CyA and internal standard peaks was achieved by using a stainless steel analytical column packed with 4 μm Nova-Pak Phenyl material. The system was operated at 75 °C using a mobile phase consisting of acetonitrile–0.04 M monobasic potassium phosphate (pH 2.5) (65:35 v/v) at a flow rate of 1 ml/min. The calibration curve for CyA in rat blood was linear over the tested concentration range of 0.0033–0.0166 M with a correlation coefficient of 0.989. For rat plasma, the range of the concentrations tested were between 0.002 and 0.0166 M and showed linearity with a correlation coefficient of 0.953. The intra- and inter-run precision and accuracy results were 1.24–21.87 and 3.1–12.23%, respectively. The low volume of blood or plasma needed (200 μl), simplicity of the extraction process, short run time (5 min) and low injection volume (30 μl) make this method suitable for quick and routine analysis.     

Comparative evaluation between capillary electrophoresis and high-performance liquid chromatography for the analysis of florfenicol in plasma

A capillary electrophoresis (CE) and a reversed phase high-performance liquid chromatography (RP-HPLC) method with UV detection have been developed for florfenicol analysis in plasma samples. The suitabilities of both methods for quantitative determination of florfenicol were approved through validation specification, such as linearity, precision, selectivity, accuracy, limit of detection and quantification. The capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) assay were compared by analyzing a series of plasma samples containing florfenicol in different concentrations using the two methods. The extraction procedure is simple and no gradient elution or derivatization is required. Furthermore, the analysis time of the CE method is two times shorter than the respective parameter in HPLC and solvent consumptions is considerably lower. The calibration curve were linear to at least 0.05–10 μg/ml (r = 0.9998) and 0.1–10 μg/ml (r = 0.9998) for CE and HPLC, respectively. The separation efficiency are good for both methods. The detection limits for florfenicol were 0.015 μg/ml with CE and 0.03 μg/ml with HPLC and CE method gave lower value, even though UV detector was applied in the both cases. The both methods were selective, robust and reliable quantification of florfenicol and can be useful for clinical and biomedical investigations.     

High-performance liquid chromatographic determination of ciprofloxacin in plasma samples

A new bioanalytical high-performance liquid chromatographic (HPLC) method for the determination of ciprofloxacin with norfloxacin as an internal standard was developed and validated for plasma samples. Norfloxacin is structural homologue of ciprofloxacin and exhibits similar retention properties. The quality of respective peak separation is strongly influenced by amphoteric character of ciprofloxacin and norfloxacin as well. In previously published HPLC methods on conventional C18 reversed-phase [F. Belal, A.A. Al-Majed, A.M. Al-Obaid, Talanta 50 (1999) 765–786; G. Carlucci, J. Chromatogr. A 812 (1998) 343–367], ion pair reagents were added into the mobile phase to suppress peak tailing. In comparison with endcapped and high purity silica reversed-phase sorbent (Purospher RP-18e, Merck), which yielded symmetrical peaks, separation efficiency was further enhanced in our method. Gradient elution mode using acetonitril and phosphate buffer pH 3 on the pentafluorophenylpropyl stationary phase (250–4.6 mm Discovery^® HS F5, 5 μm, Supelco) was carried out. The resolution of 4.1 for ciprofloxacin–norfloxacin peaks was achieved. Sample preparation by SPE C18 (Supelclean) with recovery 72% was performed. Fluorescence detection with λ_excit = 280 nm, λ_emis = 446 nm was used. After the validation, the bioanalytical HPLC method was applied to pharmacokinetic studies.     

Development of a simple, rapid and reproducible HPLC assay for the simultaneous determination of hypericins and stabilized hyperforin in commercial St. John's wort preparations

A reversed-phase HPLC method was developed and validated for the simultaneous determination of hypericins and stabilized hyperforin in St. John's Wort extract. The sample solution was prepared by extraction of the finely powdered extract with methanol–water (80:20, v/v) containing 5% HP-β-cyclodextrin, and adjusted to pH 2.5 with orthophosphoric acid. Diluted extract solutions, maintained at 0 °C, were injected into a C₁₈ column. The samples were eluted isocratically using a mobile phase consisting of acetonitrile and 0.3% v/v phosphoric acid (90:10, v/v) at a 1.5 ml/min flow rate with simultaneous fluorescence (315/590 nm, excitation/emission) and UV (273 nm) detection. Quantification of the marker compounds (hypericin, pseudohypericin, hyperforin) was achieved by use of standard curves generated by plotting peak heights versus concentrations. Validation studies demonstrated that this HPLC method is simple, rapid, reliable, and reproducible. The standard curves were linear over the concentration ranges, 0.5–2.5 μg/ml (hypericin), 0.35–1.6 μg/ml (pseudohypericin) and 5–50 μg/ml (hyperforin). The intra-day coefficients of variation obtained for hypericin, pseudohypericin and hyperforin were 4.4%, 5.4%, and 2.8%, respectively; inter-day CVs were 5.8%, 4.9%, and 2.5%, respectively. This method may be applied for the routine standardization of St. John's Wort products against hyperforin and the hypericins, the putative antidepressant principles in the herbal.     

Assay of gamma-glutamylcysteine synthetase activity in Plasmodium berghei by liquid chromatography with electrochemical detection

This work describes a high-performance liquid chromatography (HPLC) method to determine γ-glutamylcysteine (γ-GC), the intermediate product of glutathione biosynthesis. Separation relies on isocratic reversed-phase chromatography using a Symmetry C18 HPLC column, particle size 5 μm, 4.6×250 mm i.d. The mobile phase is methanol–dibasic sodium phosphate (pH 6.6; 2.8 mM) (10:90, v/v) at the flow-rate of 0.5 ml/min and detection is operated electrochemically (+200 and +550 mV) with a pre-column derivatisation reaction using ortho-phthalaldehyde (OPA) as reagent. Under these conditions the calibration range of γ-GC was 0.3–10 μg/ml; the limit of quantification was 0.3 μg/ml; accuracy, expressed as %Bias, was <10 and precision (%CV) was <6. The proposed HPLC assay was used to quantitate the γ-glutamylcysteine produced by the γ-glutamylcysteine synthetase of the rodent malaria parasite Plasmodium berghei in an in vitro enzymatic assay.     

An HPLC method for the determination of atorvastatin and its impurities in bulk drug and tablets

A simple high-performance liquid chromatographic (HPLC) method was developed for the analysis of atorvastatin (AT) and its impurities in bulk drug and tablets. This method has shown good resolution for AT, desfluoro-atorvastatin (DFAT), diastereomer-atorvastatin (DSAT), unknown impurities and formulation excipients of tablets. A gradient reverse-phase HPLC assay was used with UV detection. Some solvent systems prepared using methanol or acetonitrile and water or buffer systems with different pH values were tested. Capacity factors of related substances were calculated at all tested systems. Best resolution has been determined using a Luna C₁₈ column with acetonitrile–ammonium acetate buffer pH 4-tetrahydrofuran (THF) as mobile phase. Samples were eluted gradiently with the mobile phase at flowrate 1.0 ml min⁻¹ and detected at 248 nm. The proposed method was applied to the determination of impurities and were found to contain 0.057–0.081, 0.072–0.097, 0.608–0.664% of the DFAT, DSAT and total impurity, respectively.     

Development and validation of a sensitive liquid chromatographic method for the analysis of a novel radioprotectant: ON 01210.Na

ON 01210.Na is a chlorobenzylsulfone derivative with potential property to mitigate the effects of accidental or intentional exposure to life threatening levels of radiation. A simple and sensitive HPLC method was developed and validated for the assay of ON 01210.Na. The isocratic system used a mobile phase consisting of acetonitrile:0.1% trifluroacetic acid in water (60:40, v/v) at a flow rate of 1 ml/min. The method used a C-18 Gemini column (250 mm × 4.6 mm) with column effluents monitored at 254 nm. Forced degradation of the drug was achieved by autoclaving ON 01210.Na with 0.05N HCl, 0.05N NaOH or 1.5% (v/v) hydrogen peroxide. The assay validation parameters evaluated include specificity, linearity, precision, accuracy and sensitivity. The retention time of the drug and the other effluents were well within 7 min. Standard curves were linear over the concentration range of 10–500 μg/ml. The R.S.D. values for the within-day and day-to-day precision ranged from 0.4 to 2.5 and 2.2 to 4.4%, respectively. The R.S.D. for accuracy measurement ranged from 0.85 to 1.7%. The critical level, the detection level and the determination level for this assay were 2.86 ± 0.67, 5.69 ± 0.67 and 15.6 ± 1.8 μg/ml, respectively. A simple, sensitive and stability indicating HPLC assay was developed and validated for the analysis of a novel radioprotectant. This method was used to evaluate the aqueous as well as solid-state stability of this drug during autoclaving.     

Method development and validation for the HPLC potency assay of troglitazone tablets

This paper describes the development and validation of an isocratic, reversed-phase, high performance liquid chromatographic (HPLC) method for the assay of 200-mg troglitazone tablets. The chromatographic conditions of the method employ a YMC ODS-A, 120 A (4.6 x 150 mm, 5 microm) column, isocratic elution with (50 mM aqueous NaH2PO4, pH 4.0):acetonitrile:methanol, (35:50:15, v/v/v) as the mobile phase at a flow rate of 1.0 ml/min, a 10 microl injection volume, and ulltraviolet (UV) detection at 225 nm. The active was analyzed at ambient column temperature, using peak area responses.     

The determination of oxalic acid, oxamic acid, and oxamide in a drug substance by ion-exclusion chromatography

Oxalic acid, oxamic acid and oxamide are potential impurities in some active pharmaceutical ingredients (API). The retention and separation of oxalic and oxamic acids are particularly challenging using conventional reversed-phase HPLC due to their high polarity. An ion-exclusion chromatography (IEC) method has been shown to provide good separation and sensitivity for the three oxalate-related impurities in a hydrophobic API matrix. The method uses a Dionex IonPac ICE-ASI column with 95/5 (v/v) 0.1% sulfuric acid/acetonitrile as the mobile phase and UV detection at 205 nm. Development and validation of this method are described.