Development and validation of a rapid high-performance liquid chromatography method for the quantification of exenatide

The objective was to develop a simple HPLC method to quantify exenatide—a 39 amino acid residue incretin mimetic used in diabetes therapy. To date, only non‐validated, sometimes incomplete, gradient methods have been reported in the literature. Isocratic separation was achieved using a C₄ column and a mixed solvent system, A–B–C (48:45:7, v/v/v; pH* 5.2), where A represents KH₂PO₄ (pH 4.5; 0.1 m ) and MeCN (60:40, v/v), B corresponds to NaClO₄·H₂O (pH 6.0; 0.2 m ) and MeCN (60:40, v/v), and C is water. Exenatide eluted at 3.64 min and the total run time was 6 min. The method was specific and the response was accurate, precise and linear from 0.75 to 25 µg/mL. It was used to quantify exenatide transport across intact and laser‐porated porcine skin in vitro as a function of laser fluence [0 (i.e. intact skin), 9 and 15 J/cm², respectively]. Although no permeation was observed using intact skin, cumulative exenatide permeation after 8 h through laser porated skin was 9.6 ± 6.5 and 12.4 ± 6.4 µg/cm² at fluences of 9 and 15 J/cm², respectively. This is the first validated isocratic method for exenatide quantification and it may be of use in quality control analysis and with other biological matrices. Copyright © 2010 John Wiley & Sons, Ltd.     

Development and validation of a high-performance liquid chromatography method for quantification of egonol and homoegonol in Styrax species

Styrax camporum Pohl, known in Brazil as 'estoraque do campo' or 'cuia de brejo', has been used in the treatment of gastrointestinal diseases. The therapeutic action of S. camporum has been attributed to the ethyl acetate fraction, although the chemical composition of this fraction has not yet been analyzed. In this study, a high-performance liquid chromatography photodiode array detection (HPLC-PAD) method for analysis of Brazilian Styrax species has been developed. The compounds egonol (1) and homoegonol (2) were found to be present in all the samples investigated by HPLC. These compounds were isolated by open column chromatography followed by preparative TLC, and were identified by 1H NMR. Compounds 1 and 2 were thus proposed as phytochemical markers for Styrax, owing to their biological properties and presence in other Styrax species. The developed method has been validated and successfully applied for quantification of 1 and 2 in S. camporum dried leaves and crude ethanolic extracts from S. ferrugineus and S. pohlii aerial parts.     

高效液相色谱法定量测定中药千层塔提取物中的石杉碱甲

建立了高效液相色谱快速定量测定中药千层塔提取物中石杉碱甲含量的分析方法。千层塔提取物经甲醇/水/甲酸(10/90/0.2, v/v/v)提取并定容后,过滤膜后直接分析。色谱分离选用XCharge C18色谱柱(150 mm×4.6 mm, 5 μm),以水(含0.1%三氟乙酸)和乙腈(含0.09%三氟乙酸)为流动相进行梯度洗脱,流速为2 mL/min,于310 nm波长下检测,可在10 min内完成石杉碱甲的快速分离分析。结果表明,石杉碱甲在2.12～106 mg/L范围内线性关系良好(相关系数为0.9999);平均加标回收率为102.34%,相对标准偏差(RSD)为0.46%;日内及日间精密度均小于2%,满足定量要求。该方法简便、快速,结果可靠,重现性好,可作为千层塔提取物质量评价的依据。     

Development and validation of a sensitive quantification method for hematoporphyrin monomethyl ether in plasma using high-performance liquid chromatography with fluorescence detection

A rapid, sensitive, precise and specific method for determination of hematoporphyrin monomethyl ether (HMME), a novel photodynamic therapy (PDT) drug, was developed and validated using high-performance liquid chromatography (HPLC) with fluorescence detection. HMME was isolated from the plasma by a single-step liquid-liquid extraction with ethyl acetate. The analyte and internal standard fluorescein were baseline separated on a Diamonsil C(18) analytical column (4.6 x 150 mm, 5 microm) and analyzed using a fluorescence detector with the excitation and emission wavelengths set at 395 and 613 nm, respectively. The method was linear in the concentration range 0.025-5 microg/mL with a lower limit of quantitation (LLOQ) of 10 ng/mL. The inter- and intra-day accuracies and precisions were all within 10% and the mean recoveries of HMME and fluorescein were 95 +/- 3.7 and 90 +/- 2.3%, respectively. The analyte was stable during all sample storage, preparation and analysis periods. This method was successfully applied to a pharmacokinetic study after a single-dose intravenous administration of HMME (5 mg/kg) to beagle dogs. This method was reproducible and sensitive enough for the pharmacokinetic study of HMME. Based on the results of the pharmacokinetic study, we suggest that a rather long light-avoiding time is essential for patients under HMME therapy.     

Quantitation of tolmetin by high-performance liquid chromatography and method validation

A high-performance liquid chromatographic (HPLC) assay method for assessing the degradation of tolmetin (TLM) is developed and validated under acidic, basic, and photoirradiated conditions. The HPLC method includes an Inertsil 5 ODS-3V column (250- x 4.6-mm i.d.), guard column of Inertsil 7 ODS-3V (50- x 4.6-mm i.d.), mobile phase of CH(3)OH-1% HOAc (64:36, v/v), and UV detection at 254 nm. The developed method satisfies the system suitability criteria, peak integrity, and resolution for the parent drug and its degradants. The established assay method exhibits good selectivity and specificity suitable for stability measurements. From the intra- and interday tests of six replicates, the coefficients of variation are between 0.20% and 1.77% for the former, and 0.12% and 3.40% for the latter. Recoveries are found to be 98.7-101.7%. TLM is determined to be more reactive when exposed to light and acidic conditions, yet TLM is stable in a basic medium. A kinetic study of the photodegradation of TLM shows that it follows an apparent first-order reaction in three alcoholic solvents.     

Development and validation of a capillary high-performance liquid chromatography/electrospray tandem mass spectrometric method for the quantification of bisphenol A in air samples

Bisphenol A (BPA) is a toxic industrial chemical that affects the endocrine system even at low concentrations. A new method, based on capillary high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) analysis, has been developed to determine BPA in atmospheric samples. The method involves collection of air samples (typically 2 m(3)) on glass fiber filters, with ultrasonic extraction and sample concentration under vacuum before analysis. HPLC analysis was performed isocratically at a flow rate of 10 microL min(-1) using a capillary reversed-phase column and MS/MS analysis in negative ion multiple reaction monitoring (MRM) mode, using BPA-d(16) as internal standard. The present method provides linear response in the range 0.007-3.5 microg/filter (R(2) > 0.999) and is characterized by high accuracy (mean bias 2%) and good reproducibility (mean RSD 5%). High sensitivity (LOD = 2 ng/m(3) based on 2 m(3) of air collected), specificity, and speed of the analysis make the present method suitable for routine determination of BPA in the atmosphere, both for ambient and personnel monitoring.     

A high-performance liquid chromatographic method for saikosaponin a quantification in rat plasma

A high-performance liquid chromatographic method with UV detection has been developed for the determination of saikosaponin a in rat plasma. Saikosaponin a and internal standard jujuboside A were isolated from plasma samples by solid-phase extraction. The chromatographic separation was achieved on a reversed-phase C(18) column with the mobile phase of acetonitrile-water (35:65, v/v) at a flow rate of 1 mL/min and UV detection was set at 205 nm. The standard curve for saikosaponin a was linear over the concentration range 0.25-10 microg/mL and the limit of detection was 0.05 microg/mL. The absolute recovery was greater than 82%. The precision and accuracy ranged from 3.05 to 9.59% and 95.61 to 110.00%, respectively. The validated method was used to determine saikosaponin a in plasma samples in a pharmacokinetic study of saikosaponin a administered to Sprague-Dawley rats.     

Development and validation of a high-performance liquid chromatography-tandem mass spectrometric method for quantification of daunorubicin in rat plasma

A high-performance liquid chromatography-tandem mass spectrometric method (LC-MS/MS) has been developed and validated for the determination of daunorubicin in K₃EDTA rat plasma. The 100 μL plasma samples were extracted by a methanol:acetone protein precipitation step in the presence of additional 50 μL of 70% (w/v) zinc sulfate, and subsequently analyzed by LC/MS/MS using positive turbo-ion spray ionization mode. The LC/MS/MS instrument was operated in the multiple-reaction-monitoring (MRM) mode. Doxorubicinol was better than doxorubicin as the internal standard because its recovery and absolute matrix effect data exactly matched with those for daunorubicin. In addition, HPLC gradient condition was optimized to thoroughly separate daunorubicin from the background interference. The validated concentration range was from 0.250 to 100 ng/mL. The true recoveries of daunorubicin and doxorubicinol were 93.2% and 93.6%, respectively. In addition, the ion-suppression data of daunorubicin and doxorubicinol were 78.2% and 78.4%, respectively. Absence of the relative matrix effect from six unique lots was confirmed. Results obtained from the GLP validation study demonstrated very good accuracy (95-105%) and precision (less than 10% CV).     

A simple method for nicardipine hydrochloride quantification in plasma using solid-phase extraction and reversed-phase high-performance liquid chromatography

A simple and sensitive reversed-phase liquid chromatography method was developed and validated for the determination of nicardipine hydrochloride (NC) in rabbit plasma. Nicardipine hydrochloride and nimodipine, used as internal standard, were initially extracted from plasma by a rapid solid-phase extraction using C(18) cartridges. After extraction, nicardipine hydrochloride was separated by HPLC on a C(18) column and quantified by ultraviolet detection at 254 nm. A mixture of acetonitrile-0.02 M sodium phosphate buffer-methanol (45:40:15) with 0.2% of triethylamine of pH of 6.1 was used as mobile phase. The mean (+/-SD) extraction efficiency of NC was 77.56 +/- 5.4, 84.23 +/- 4.32 and 83.94 +/- 3.87% for drug concentrations of 5, 25 and 100 ng/mL, respectively. The method proved to be linear in the range of 5-100 ng/mL with a regression coefficient of 0.9993. The relative standard deviations of intra- and inter-day analysis for NC in plasma were 3.26-6.52% (n = 5) and 4.71-9.38% (n = 5), respectively. The differences of the mean value measured from the concentration prepared, expressed in percentages (bias percentage), were only - 5.2, 0.4 and 0.8% at NC 5, 25 and 50 ng/mL, which confirmed the accuracy of the method. The analytical technique was used to determine NC plasma concentration after drug oral administration to rabbits. The results inferred that NC is rapidly absorbed in rabbits and has a short half-life (t(1/2) = 1.34 h).     

Development and validation of a column high-performance liquid chromatography method for quantification of ganaphaliin A and B in inflorescences of Gnaphalium liebmannii Sch. Bp ex Klatt

An HPLC method was developed for the simultaneous determination of gnaphaliin A and B, active compounds of Gnaphalium liebmannii Sch. Bp ex Klatt. The HPLC separation was performed on an Inertsil ODS-3 (150 x 4.6 mm id, 5 microm) RP C18 column operated at 40 degrees C; the isocratic mobile phase was 0.02% aqueous orthophosphoric acid-methanol-acetonitrile (50 + 30 + 20, v/v/v), with a run time of 20 min and flow rate of 1.5 mL/min. Detection with a photodiode array detector (PDAD) was at 270 nm. The method was validated for linearity, repeatability, LOD, and LOQ. The LOD and LOQ for gnaphaliin A and B were found to be in the range of 0.4-0.5 and 1.0-1.4 microg/mL, respectively. This is the first report of an analytical method developed for the quantitative analysis of flavones from Gnaphalium species by HPLC-PDAD with applications for raw material and commercial products.     

Development and validation of high-performance liquid chromatography method for determination of clarithromycin in pharmaceutical tablets

Clarithromycin is a very important macrolide antibiotic used to treat bacterial infections in human and veterinary medicine. This study reports the development and validation of cost-effective, simple, precise, accurate, and robust high-performance liquid chromatography (HPLC) for the determination of clarithromycin (CLA) in tablets. Reversed-phase chromatography was conducted using a standard column at 55°C with ultraviolet detection at 215 nm. A mobile phase consisting of acetonitrile –2-methyl-2-propanol –potassium phosphate buffer was used at a flow rate of 1.0 mL/min. The proposed method displayed good linearity, precision, accuracy, robustness, and specificity. The present HPLC was compared with capillary electrophoresis and bioassay methods and the results indicated that there was no significant difference between these methods. Moreover, the obtained results demonstrated the validity of the isocratic HPLC, which allows reliable quantitation of CLA in pharmaceutical samples. Thus, it can be used as a substitute alternative methodology for the routine quality control of this medicine, in situations where other methods are less accessible in the laboratory.     

Development,method validation and simultaneous quantification of eleven bioactive natural products from high-altitude medicinal plant by high-performance liquid chromatography

Herein, a novel, rapid, reliable, simple method validation and simultaneous quantification of 11 bioactive compounds (mostly xanthones) have been described. International Conference on Harmonization guidelines were used for the analytical method validation. Good linearity, repeatability, intra- and inter-day precision, accuracy and reliability were well-illuminated in the method validation procedure. The calibration curves showed a good linear relationship (r > 0.999) within test range. Precision was evaluated by intra- and inter-day tests with relative standard deviation <2.79% and accuracy validation recovery of 74.16%–91.84%. On quantification study, the validated method described the high content of bioactive xanthone derivatives, including 1-hydroxy-3, 5-dimethoxyxanthone ( 7 ), 2-(allyloxy)-8-hydroxy-1, 6-dimethoxyxanthone ( 6 ) 1, 7, 8-trihydroxy-3-methoxyxanthone ( 9 ) and Coxanthone E ( 5 ) in Codonopsis ovata, which is advantageous given the numerous pharmacological and biological effects associated with these compounds, which mostly exhibit anti-cancerous, antioxidant, anti-inflammatory, anti-mutagenic and anti-obesity effects. The bulk abundance of these compounds can also be used for further modification to produce better lead molecules for drug discovery with low toxicity and high potency. The proposed method makes it possible to simultaneously determine all bioactive compounds in one run and can be extended to marker-based standardization of herbal formulations in medicinal and pharmaceutical industries.     

Validation of a new reversed-phase high-performance liquid chromatography method for separation and quantification of bovine milk protein genetic variants

A new RP-HPLC method for the separation and quantification of the most common genetic variants of bovine milk proteins is described. A reversed-phase analytical column C8 (Zorbax 300SB-C8 RP, 3.5 microm, 300A, 150 x 4.6 I.D.) was used. All the most common casein (CN) and whey protein genetic variants, including beta-CN(I) were detected and separated simultaneously in less then 40 min, with the exception of alpha(S1)-CN(B) and CN(C) variants. Purified protein genetic variants were employed in calibration and showed different absorbances at 214 nm. The procedure was developed using 40 raw individual milk samples of cows belonging to four different breeds and certified skim milk powder BCR-063R. Method validation consisted in testing linearity, repeatability, reproducibility and accuracy. A linear relationship (R(2)>0.99) between the concentrations of proteins and peak areas was observed over the concentration range, with low detection limits. Repeatability and reproducibility were satisfactory for both retention times and peak areas. The RSD of peak areas ranged from 0.92 to 4.32% within analytical day and from 0.85 to 9.52% across analytical days. The recoveries, calculated using mixtures of samples previously quantified, ranged from 98.1 to 103.7%.     

Development and validation of a new high-performance liquid chromatography method for the determination of gliclazide and repaglinide in pharmaceutical formulations

A new high-performance liquid chromatography method was developed and validated for the quantitation of gliclazide and repaglinide in pharmaceutical formulations. Determination was performed using a LiChroCART RP-18 column, a mobile phase containing acetonitrile-phosphate buffer (pH 2.1; 60 + 40, v/v), and ultraviolet (UV) detection at 225 nm. Repaglinide was used as an internal standard for gliclazide determination and gliclazide for repaglinide assay. The method was validated with respect to linearity, precision, robustness, ruggedness, accuracy, and specificity. The calibration graphs ranged from 0.015 to 0.09 mg/mL for gliclazide and 0.06 to 0.36 mg/mL for repaglinide. Intra- and interday relative standard deviation values for the standard solutions were 0.70 and 1.01% for gliclazide and 0.78 and 0.93% for repaglinide, respectively. Total recoveries of gliclazide and repaglinide from the laboratory-prepared mixtures were 99.82 +/- 0.58 and 101.50 +/- 0.46% for gliclazide and repaglinide, respectively [mean +/- standard deviation (SD)]. In forced degradation studies, the effect of acid, base, oxidation, UV light, and temperature on both drugs was also investigated. Finally, the method was applied for the quality control of commercial gliclazide and repaglinide tablets. Total recovery was 100.40 +/- 0.35 and 104.46 +/- 0.23% for gliclazide and repaglinide, respectively (mean +/- SD).     

Optimization and validation of a high-performance liquid chromatography method for the analysis of cardiac glycosides in Digitalis lanata

In this study, a simple and reliable HPLC method for the qualitative and quantitative analysis of cardiac glycosides in Digitalis lanata Ehrh. raw material was developed and applied to healthy and phytoplasma-infected plants. The target analytes cover a broad range of secondary metabolites, including primary, secondary and tertiary glycosides and the corresponding aglycones. The sample preparation was carried out by sonication of the plant material with 70% (v/v) aqueous methanol at room temperature, followed by reversed-phase solid-phase extraction purification from interfering pigments. The HPLC analyses were performed on a Symmetry C₁₈ column (75 mm × 4.6 mm I.D., 3.5 μm), with a gradient elution composed of water and acetonitrile, at a flow rate of 1.0 mL/min. The column temperature was set at 20 °C and the photodiode array detector monitored the eluent at 220 nm. The method was validated with respect to ICH guidelines and the validation parameters were found to be highly satisfactory. The application of the method to the analysis of D. lanata leaves indicated that air-drying was the optimum method for raw material processing when compared with freeze-drying. The analysis of healthy and phytoplasma-infected plants demonstrated that the secondary metabolite mainly affected by the pathogen presence was lanatoside C (153.2 μg/100 mg versus 76.1 μg/100 mg). Considering the importance of D. lanata plant material as source of cardiac glycosides, the developed method can be considered suitable for the phytochemical analysis and for the quality assurance of D. lanata used for pharmaceutical purpose.     

Quick bioanalytical method of quantification and validation of sulbactam in plasma using high performance liquid chromatography

A quick method of quantitative determination of sulbactam in human plasma, using liquid chromatography-UV spectroscopy, has been developed and validated. After derivatization with imidazole, plasma samples were treated by direct deproteinization with acetonitrile as an extraction solvent. After ultracentrifugation, sulbactam extract was directly injected onto the LC column. Chromatographic separation was performed on TSK Gel Super ODS (50 mm × 4.6 mm i.d., 2 μm) using methanol and phosphate buffer with tetrabutylammonium hydroxide solution as a mobile phase. Gradient elution was employed. The method was fully validated according to the United States Food and Drug Administration requirements (linearity, precision, trueness, quantification limit, detection limit, recovery, specificity and stability). The calibration curves were linear within the concentration range of 0.05–4.0 μg mL⁻¹. Good method/system precision and accuracy of the method were demonstrated.      

An improved method for high-performance liquid chromatography of gossypol

Earlier liquid-chromatographic methods for the determination of gossypol, based on highly acidic methanolic solvents, provide broad tailing peaks. The use of acetonitrile with aqueous phosphoric acid/tri-n-butylammonium phosphate at pH 3.5 and a high-resolution radial-compression column gave greatly improved performance and excellent peak shape.     

化学计量学二阶校正方法结合高效液相色谱用于蜂蜜中10种酚酸类物质的快速定量分析

利用化学计量学二阶校正方法结合高效液相色谱对枣花蜜中10种酚酸类物质的快速定量分析进行了研究。首先通过验证样本研究了所建立模型的准确性。结果显示:10种酚酸类物质的线性相关系数(R)为0.998 2～0.999 9,平均回收率为97.6%～101.1%,说明所建立的模型稳定可靠。其次,通过模拟蜂蜜试验,确定了固相萃取柱的种类及操作条件(HLB柱,酸化水淋洗,甲醇洗脱)。最后,利用模拟蜂蜜得到的最优条件结合化学计量学二阶校正方法,测定了枣花蜜中10种酚酸类物质的含量,并测得其加标回收率为62.1%～93.8%,考虑到目标分析物的种类较多,且蜂蜜基质极为复杂,该结果基本满足要求。另外,还利用统计与品质因子验证了试验方法的可靠性,结果令人满意。该方法具有简单、快速等优点,可用于复杂基质中多种目标分析物的同时定量分析。