Quantitative determination and sampling of azathioprine residues for cleaning validation in production area

Cleaning validation is an integral part of current good manufacturing practices in any pharmaceutical industry. Nowadays, azathioprine and several other pharmacologically potent pharmaceuticals are manufactured in same production area. Carefully designed cleaning validation and its evaluation can ensure that residues of azathioprine will not carry over and cross contaminate the subsequent product. The aim of this study was to validate simple analytical method for verification of residual azathioprine in equipments used in the production area and to confirm efficiency of cleaning procedure. The HPLC method was validated on a LC system using Nova-Pak C18 (3.9 mm x 150 mm, 4 microm) and methanol-water-acetic acid (20:80:1, v/v/v) as mobile phase at a flow rate of 1.0 mL min(-1). UV detection was made at 280 nm. The calibration curve was linear over a concentration range from 2.0 to 22.0 microg mL(-1) with a correlation coefficient of 0.9998. The detection limit (DL) and quantitation limit (QL) were 0.09 and 0.29 microg mL(-1), respectively. The intra-day and inter-day precision expressed as relative standard deviation (R.S.D.) were below 2.0%. The mean recovery of method was 99.19%. The mean extraction-recovery from manufacturing equipments was 83.5%. The developed UV spectrophotometric method could only be used as limit method to qualify or reject cleaning procedure in production area. Nevertheless, the simplicity of spectrophotometric method makes it useful for routine analysis of azathioprine residues on cleaned surface and as an alternative to proposed HPLC method.     

Development and validation of a UPLC method for quality control of rhubarb-based medicine: fast simultaneous determination of five anthraquinone derivatives

A reverse phase ultra performance liquid chromatography (UPLC) method was developed for the rapid quantification of five anthraquinone derivatives (aloe-emodin, rhein, emodin, chrysophanol and physcion) in rhubarb using a Waters Acquity BEH C18, 50 mm x 2.1 mm, 1.7 microm column. The runtime was as short as 3 min. The influence of flow rate and column temperature on resolution was investigated. The method was validated according to the regulatory guidelines with respect to precision, accuracy, linearity and robustness. Comparison of system performance with conventional HPLC was made with respect to analysis time, efficiency and sensitivity. The proposed method was found to be reproducible and convenient for quantitative analysis of five anthraquinone derivatives in three species of rhubarb and related preparations.     

Development and validation of UPLC method for determination of primaquine phosphate and its impurities

With the objective of reducing analysis time and maintaining good efficiency, there has been substantial focus on high-speed chromatographic separations. Recently, commercially available ultra-performance liquid chromatography (UPLC) has proven to be one of the most promising developments in the area of fast chromatographic separations. In this work, a new isocratic reverse phase chromatographic method was developed using UPLC for primaquine phosphate bulk drug. The newly developed method is applicable for assay and related substance determination of the active pharmaceutical ingredient. The chromatographic separation of primaquine and impurities was achieved on a Waters Acquity BEH C18, 50 x 2.1mm, 1.7 microm column within a short runtime of 5 min. The method was validated according to the regulatory guidelines with respect to specificity, precision, accuracy, linearity and robustness. Forced degradation studies were also performed for primaquine phosphate bulk drug samples to demonstrate the stability indicating power of the UPLC method. Comparison of system performance with conventional HPLC was made with respect to analysis time, efficiency and sensitivity.     

埃索美拉唑钠中间体清洁验证残留限度检查方法学研究

目的：埃索美拉唑钠合成中间体清洁验证残留限度检查方法学研究。方法：色谱柱为Agilent ZORBAX SB-C18,4.6mm×150mm,5μm;以乙腈-磷酸盐缓冲液（pH7.6）（50∶50）为流动相;流速1.0mL·min-1;检测波长302nm;进样量10μL。结果：中间体的线性范围为0.06μg·mL-1～1.5μg·mL-1;定量限和检出限分别为0.24ng和0.08ng;平均回收率为89.29%,RSD为1.8%。结论：本方法简便、快速、准确、精密度高、回收率好,可用于中间体的清洁验证。     

Development and validation of a stability indicating UPLC method for determination of ticlopidine hydrochloride in its tablet formulation

The objective of the current study was the development of a simple, precise and accurate isocratic reversed-phase stability indicating Ultra Performance Liquid Chromatography [UPLC] assay method and validated for determination of ticlopidine hydrochloride in solid pharmaceutical dosage forms. Isocratic separation was achieved on a Zorbax SB-C18 (50 mm × 4.6 mm, 1.8 μm) column using mobile phase of methanol–0.01 M ammonium acetate buffer, pH 5.0 (80:20, v/v) at a flow rate of 0.8 ml min⁻¹, the injection volume was 4.0 μl and the detection was carried out at 235 nm by using photo-diode array detector. The drug was subjected to oxidation, hydrolysis, photolysis and heat to apply stress condition. The method was validated for specificity, linearity, precision, accuracy, robustness and solution stability. The method was linear in the drug concentration range of 62.5–375 μg ml⁻¹ with a correlation coefficient of 0.9999. The precision (relative standard deviation – RSD) of six samples was 1.31% for repeatability and the intermediate precision [RSD] among six-sample preparation was 0.77%. The accuracy (recovery) was between 98.80% and 101.50%. Degradation products produced as a result of stress studies did not interfere with detection of ticlopidine hydrochloride and the assay can thus be considered stability indicating.     

Development and validation of HPLC method for the determination of pregabalin in capsules

A simple, precise, specific, and accurate reverse phase HPLC method has been developed for the determination of pregabalin in capsule dosage form. The chromatography was set on Hypersil BDS, C8, 150×4.6 mm, 5 μm column using photodiode array detector. The mobile phase consisting of phosphate buffer pH 6.9 and acetonitrile in the ratio of 95:05 with flow rate of 1 ml/min. The method was validated according to ICH guidelines with respect to specificity, linearity, accuracy, precision and robustness. Lower limit of quantification is 0.6 mg/l. The pregabalin sample solution was found to be stable at room temperature for about 26 h.     

Development and validation of UPLC method for quality control of Curcuma longa Linn.: Fast simultaneous quantitation of three curcuminoids

A new reversed phase ultra performance liquid chromatography (UPLC) method was developed for the rapid quantification of three curcuminoids (curcumin (C), desmethoxycurcumin (DMC) and bisdesmethoxycurcumin (BDMC)) in Curcuma longa Linn. (C. longa) using a Waters BEH Shield RP C₁₈, 2.1 mm × 100 mm, 1.7 μm column. The runtime was 2 min. The influence of column temperature and mobile phase on resolution was investigated. The method was validated according to the ICH guideline for validation of analytical procedures with respect to precision, accuracy, and linearity. The limits of detection were 40.66, 49.38 and 29.28 pg for C, DMC and BDMC, respectively. Limits of quantitation for C, DMC and BDMC, were 134.18, 164.44 and 97.50 pg, respectively. Linear range was from 3.28 to 46.08 μg/ml. The mean ± SD percent recoveries of curcuminoids were 99.47 ± 1.66, 99.50 ± 1.99 and 97.77 ± 2.37 of C, DMC and BDMC, respectively. Comparison of system performance with conventional HPLC was made with respect to analysis time, efficiency and sensitivity. The proposed method was found to be reproducible and convenient for quantitative analysis of three curcuminoids in C. longa. This work provided some references for quality control of C. longa.     

Development and validation of an ion chromatography method for the determination of phosphate-binding of lanthanum carbonate

Lanthanum carbonate is indicated to reduce serum phosphate in patients with end stage renal disease (ESRD). When given orally, lanthanum carbonate dissociates in the acid environment of the upper gastrointestinal tract to release lanthanum ions. The free lanthanum ions bind with dietary phosphate released from food during digestion to form highly insoluble lanthanum–phosphate complexes which prevent the absorption of phosphate, consequently reduce the serum phosphate. In order to evaluate the in vitro binding capacity of lanthanum carbonate, a simple and efficient ion chromatography (IC) method was developed and validated for determination of phosphate across the pH range encountered in the gastrointestinal tract. Chromatographic separation was achieved on a Dionex ICS-2000 IC system using a Dionex AS16, IonPac (4 mm × 250 mm) analytical column and Dionex AG16, IonPac (4 mm × 50 mm) guard column. Column temperature was maintained at 30 °C. Injection volume was 10 μL. The compounds were eluted isocratically at a flow rate of 1 mL/min and detected by suppressed conductivity. The analytical method was validated according to USP Category I requirements. The validation characteristics included accuracy, precision, quantification limit, linearity, and stability. The intra-day accuracy ranged from 89% to 103% for the solutions of pH 1.2–6.8. The intra-day precision (RSD) ranged from 0.6% to 3.7% for the solutions of pH 1.2–6.8. The analytical range was linear from 2 to 200 ppm (mg/L). The R² ranged from 0.9998 to 1.0. This method was found to be simple, robust, sensitive, specific, and accurate. It has been successfully applied for determination of phosphate binding to lanthanum carbonate over the human gastrointestinal pH range at different time-points (from 0.5 to 24 h).     

超高效液相色谱法测定阿司匹林和水杨酸的血药浓度

目的：建立并验证超高效液相色谱法（UPLC）测定人血浆中阿司匹林（ASP）和水杨酸（SA）的浓度,并应用于临床ASP和SA血药浓度的测定。方法：色谱柱为ACQUITYUPLC^® BEH C₁₈（2.1 mm×100 mm,1.7 μm）,流动相为含0.05%三氟乙酸（TFA）的超纯水和乙腈（78%：22%）,流速0.5 mL·min^-1,柱温30℃,进样器4℃,检测波长304 nm和277 nm,内标为替硝唑。结果：ASP和SA分别在0.4~100 μg·mL^-1和0.2~50 μg·mL^-1线性关系良好（r²>0.999）;ASP和SA的日内、日间准确度和精密度均<15%。结论：本研究建立了一个准确、灵敏且稳定的UPLC法测定ASP和SA血药浓度的方法,可应用于临床ASP和SA血药浓度的测定和药动学研究,指导临床合理用药。     

The use of inductively coupled plasma-atomic emission spectroscopy (ICP-AES) in the determination of lithium in cleaning validation swabs

The pharmaceutical industry is required to perform cleaning validation studies to verify that equipment used in the manufacture of pharmaceuticals is adequately cleaned from one product or process to the next. Typically, these cleaning validation studies require an analytical method that uses some form of chromatographic technique. In the case of products that may have an inorganic constituent, however, if can often be easier to verify the cleanliness of equipment by using a non-chromatographic technique. A method is described to certify the cleanliness of processing equipment by determining lithium in cleaning validation swabs using inductively coupled plasma-atomic emission spectroscopy (ICP-AES).     

Development and validation of ultra-performance liquid chromatography method for the determination of meloxicam and its impurities in active pharmaceutical ingredients

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Development and validation of an ultra performance liquid chromatography method for venlafaxine hydrochloride in bulk and capsule dosage form

A simple, specific, accurate, and precise ultra performance liquid chromatographic method was developed and validated for the estimation of venlafaxine hydrochloride in tablet dosage forms. A acquity TM BEH column having C18, 100×2.1 mm i.d. in isocratic mode, with mobile phase containing dipotassium hydrogen phosphate: Acetonitrile (30:70 v/v; pH 7.00 with dilute o-phosphoric acid) was used. The flow rate was 0.75 ml/min and effluents were monitored at 227 nm. The retention time of venlafaxine hydrochloride was 0.548 min. The method was validated for specificity, linearity, accuracy, precision, limit of quantification, limit of detection, robustness and solution stability. Limit of detection and limit of quantification for estimation of venlafaxine hydrochloride were found to be 6.11 μg/ml and 20.33 μg/ml, respectively. Recoveries of venlafaxine hydrochloride in tablet formulations were found to be in the range of 99.3-99.5%. Proposed method was successfully applied for the quantitative determination of venlafaxine hydrochloride in tablet dosage forms.     

Validation of an HPLC-UV method for the determination of ceftriaxone sodium residues on stainless steel surface of pharmaceutical manufacturing equipments

In pharmaceutical industry, an important step consists in the removal of possible drug residues from the involved equipments and areas. The cleaning procedures must be validated and methods to determine trace amounts of drugs have, therefore, to be considered with special attention. An HPLC-UV method for the determination of ceftriaxone sodium residues on stainless steel surface was developed and validated in order to control a cleaning procedure. Cotton swabs, moistened with extraction solution (50% water and 50% mobile phase), were used to remove any residues of drugs from stainless steel surfaces, and give recoveries of 91.12, 93.8 and 98.7% for three concentration levels. The precision of the results, reported as the relative standard deviation (RSD), were below 1.5%. The method was validated over a concentration range of 1.15-6.92 μg ml(-1). Low quantities of drug residues were determined by HPLC-UV using a Hypersil ODS 5 μm (250×4.6 mm) at 50 °C with an acetonitrile:water:pH 7:pH 5 (39-55-5.5-0.5) mobile phase at flow rate of 1.5 ml min(-1), an injection volume of 20 μl and were detected at 254 nm. A simple, selective and sensitive HPLC-UV assay for the determination of ceftriaxone sodium residues on stainless steel surfaces was developed, validated and applied.     

Cleaning validation 2: development and validation of an ion chromatographic method for the detection of traces of CIP-100 detergent

A cleaning validation method, ion chromatographic method with conductivity detection was developed and validated for the determination of traces of a clean-in-place (CIP) detergent. It was shown to be linear with a squared correlation coefficient (r(2)) of 0.9999 and average recoveries of 71.4% (area response factor) from stainless steel surfaces and 101% from cotton. The repeatability was found to be 2.17% and an intermediate precision of 1.88% across the range. The method was also shown to be sensitive with a detection limit (DL) of 0.13 ppm and a quantitation limit (QL) of 0.39 ppm for EDTA, which translates to less than 1 microL of CIP diluted in 100mL of diluent in both cases. The EDTA signal was well resolved from typical ions encountered in water samples or any other interference presented from swabs and surfaces. The method could be applied to cleaning validation samples. The validated method could be included as a suitable one for rapid and reliable cleaning validation program.     

Development and validation of a stability indicating RP-UPLC method for determination of quetiapine in pharmaceutical dosage form

The present work reports a stability indicating reversed phase ultra performance liquid chromatography (RP-UPLC) method for the quantitative determination of quetiapine in pharmaceutical dosage form. The chromatographic separation is performed on an Agilent Eclipse Plus C18, RRHD 1.8 μm (50 mm x 2.1 mm) column using gradient elution. The optimized mobile phase consists of 0.1 % aqueous triethylamine (pH 7.2) as a solvent-A and 80:20 v/v mixture of acetonitrile and methanol as solvent-B. The eluted compounds are monitored at 252 nm wavelength using a UV detector. The developed method separates quetiapine from its five impurities/degradation products within a run time of 5 min. Stability indicating capability of the developed method is established by analyzing forced degradation samples in which the spectral purity of quetiapine is ascertained along with the separation of degradation products from analyte peak. The developed RP-UPLC method is validated as per International Conference on Harmonization (ICH) guidelines with respect to system suitability, specificity, precision, accuracy, linearity, robustness and filter compatibility.     

Impurity profile study of lopinavir and validation of HPLC method for the determination of related substances in lopinavir drug substance

Several related substances (RS4–RS10) were detected in lopinavir drug substance at levels ranging from 0.03% to 0.1% by employing gradient RP-HPLC. The related substances were identified by LC–MS analysis. These related substances were isolated and characterized by Mass, ¹H NMR and FT-IR spectral data. The separation was achieved on a YMC Pack ODS-AQ (250 mm × 4.6 mm, 5 μm) column thermostated at 45 °C using 0.02 M KH₂PO₄ (pH 2.5): acetonitrile as a mobile phase in gradient elution mode. A PDA detector set at 210 nm was used for detection. The investigated validation elements showed the method has acceptable specificity, accuracy, linearity, precision, robustness and high sensitivity with detection limits and quantitation limits ranging from 0.028 μg/ml to 0.063 μg/ml and 0.084 μg/ml to 0.192 μg/ml respectively. The method can be used for routine quality control analysis and stability testing of lopinavir drug substance.     

Development of a stability-indicating UPLC method for determination of isotretinoin in bulk drug

A highly sensitive and rapid stability indicating ultra-performance liquid chromatographic (UPLC) method was developed for the quantification and identification of isotretinoin in bulk. Chromatographic separation was developed using a gradient elution in a reversed-phase system at flow rate of 0.5?ml/min with 12?min run time. The mobile phase was a gradient mixture of mobile phase A (contained a 30:70:0.5 mixture solution of methanol/purified water/glacial acetic acid) and mobile phase B (contained a 70:25:4.5:0.5 mixture solution of methanol/acetonitrile/purified water/glacial acetic acid). Eluents were monitored at 355?nm. The analytical method was validated for accuracy, precision, robustness, linearity, and forced degradation in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) topic Q2 (R1) ‘Validation of Analytical Procedures: Text and Methodology’. The method was linear over a concentration range of (1–7?µg/ml) with correlation coefficient of (r²?>?0.9999). The accuracy was confirmed by calculating the % recovery which was found to be 100.0–101.6%. The RSD values obtained for repeatability and intermediate precision experiments were less than 2%. The limit of detection (LOD) was 0.12?µg/ml, while the limit of quantification (LOQ) was 0.38?µg/ml. The drug samples were exposed to different stressed conditions and the results showed that all degradation products were satisfactorily separated from each other and from the peak of the drug using the developed method. The proposed method can be used for the quantitative determination of isotretinoin with confidence.     

Development and validation of rapid ion-chromatographic method with conductivity detection for trace level determination of allylamine in sevelamer drug substances

A sensitive and rapid ion chromatography (IC) method was developed for the low level determination of allylamine (AAM) in sevelamer (SVM) drug substances, i.e., sevelamer hydrochloride (SVH) and sevelamer carbonate (SVC). This method utilized a Dionex Ion Pack CS14 IC column, a mobile phase of 10 mM methane sulfonic acid with conductivity detection. The total chromatographic run time was as short as 8 min. The various factors involved in the sample preparation such as, extraction solvent, extraction time and stirrer speed were evaluated. This method was validated as per United States Pharmacopoeia (USP) and International Conference on Harmonization (ICH) guidelines in terms of detection limit, quantitation limit, linearity, precision, accuracy, specificity and robustness. Linearity of the method was very good over the concentration range of 9–750 μg/mL with the coefficient of determination (r²) 0.999. The detection and quantitation limit of AAM were 2.7 and 9.0 μg/mL, respectively. The recovery data obtained for AAM were between 97% and 109%. Also, the specificity of the method was proved through IC coupled with mass spectrometer (IC–MS). The developed method was found to be robust and applied successfully to determine the content of AAM in Sevelamer bulk drugs.     

Novel Validated Stability-Indicating UPLC Method for the Estimation of Naproxen and its Impurities in Bulk Drugs and Pharmaceutical Dosage Form

A novel, reversed-phase ultra-performance liquid chromatographic method was developed and validated for the determination of related substances in Naproxen (NAP) bulk drugs and dosage forms. The related substances included degradation and process-related impurities. The method was developed using the Waters Acquity BEH C18 column using the gradient program with mobile phase A of a pH 7.0 phosphate buffer and methanol in the ratio of 90: 10 (v/v) and mobile phase B as methanol and acetonitrile in the ratio of 50:50 (v/v). Naproxen and its impurities were monitored at 260 nm. Naproxen was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal, humidity, and photolytic degradations. The degradation products were well-resolved from the main peak and its impurities, proving the stability-indicating power of the method. The performance of the method was validated according to the present ICH guidelines for specificity, limit of detection, limit of quantification, linearity, accuracy, precision, ruggedness, and robustness.     

Development and validation of a simple HPLC method for simultaneous in vitro determination of amoxicillin and metronidazole at single wavelength

A simple, rapid, sensitive and robust reversed phase-HPLC method was developed and validated to measure simultaneously the amount of amoxicillin and metronidazole at single wavelength (254 nm) in order to assess drug release profiles and drug-excipients compatibility studies for a new floating-sustained release tablet formulation and its subsequent stability studies. An isocratic elution of filtered sample was performed on C18 column with buffered mobile phase (pH 4.0) and UV detection at 254 nm. Quantification was achieved with reference to the external standards. The linearity for concentrations between 0.15 and 600 microg/ml for amoxicillin and 0.13 and 300 microg/ml for metronidazole were established. Intra and inter-day precision were less than 2.5%. The limits of detection (LOD) and quantification were 0.05 and 0.15 microg/ml for amoxicillin and 0.10 and 0.13 microg/ml for metronidazole. The determination of the two active ingredients was not interfered by the excipients of the products. Samples were stable in the release media (37 degrees C) and the HPLC injector at least for 12 h.