Validated UPLC method for the determination of guaiphenesin,oxeladin citrate,diphenhydramine, and sodium benzoate in their quaternary mixture used in treatment of cough,in the presence of guaiphenesin-related substance (guaiacol)

Accurate, precise, and sensitive UPLC method was developed and validated for the simultaneous determination of a quaternary mixture containing guaiphenesin (GUP), oxeladin citrate (OXC), diphenhydramine (DPH), and sodium benzoate (SOB) in the presence of GUP-related substance, guaiacol (GUA). Chromatographic separation was achieved using NUCLEOSHELL^® column—C₁₈ (4.0?×?50.0 mm, 2.7 μm). Isocratic elution at a flow rate 1.0 mL/min was performed using a mobile phase consisting of acetonitrile:phosphate buffer pH 3.5?±?0.1 (25.0:75.0, v/v). The specificity of the developed method was investigated by analyzing of laboratory-prepared mixtures containing different ratios of the four drugs, in the presence of GUA as well as their combined pharmaceutical formulation. The obtained results were statistically compared with those obtained by the official or reported methods, showing no significant difference with respect to accuracy and precision at p?=?0.05.     

A validated stability‐indicating ultra performance liquid chromatography method for the determination of potential process‐related impurities in eplerenone

A simple, sensitive, and accurate stability‐indicating analytical method has been developed and validated using ultra high performance liquid chromatography. The developed method is used to evaluate the related substances of eplerenone (EP). The degradation behavior of EP under stress conditions was determined, and the major degradants were identified by ultra high performance liquid chromatography with tandem mass spectrometry. The chromatographic conditions were optimized using an impurity‐spiked solution, and the samples, generated from forced degradation studies. The resolution of EP, its potential impurities, and its degradation products was performed on a Waters UPLC BEH C₁₈ column (50 × 2.1 mm, 1.7 μm) by linear gradient elution using a mobile phase consisting of 10 mmol/L ammonium acetate adjusted to pH 4.5, methanol and acetonitrile. A photo‐diode array detector set at 245 nm was used for detection. The flow rate was set at 0.3 mL/min. The procedure had good specificity, linearity (0.02–3.14 μg/mL), recovery (96.1–103.9%), limit of detection (0.01–0.02 μg/mL), limit of quantitation (0.03–0.05 μg/mL), and robustness. The correction factors of the process‐related substances were calculated.     

A comparative study of HPLC-UV and UPLC-DAD methods for simultaneous estimation of aspirin and cilostazol in the presence of their related impurities in bulk and capsules

Aspirin (ASP) and cilostazol (CST) are used as a combination in pharmaceutical formulations for treatment of strokes. Salicylic acid (SAL) is considered to be one of the main synthesis impurities and a degradation product of ASP. On the other hand, the main related impurities of CST are CST related A, B, and C (CST-RA, CST-RB, and CST-RC), respectively. Furthermore, as high efficiency and less elution are the basic requirements of high-speed chromatographic separation, so, a comparative study of two simple, precise, and accurate reversed-phase HPLC and UPLC methods was developed and validated for simultaneous estimation of ASP and CST in bulk and capsules in the presence of SAL, CST-RA, CST-RB, and CST-RC. A Eurospher II C18 (250?×?4.6?mm², 5?µm) for HPLC method and an Agilent Zorbax Eclipse Plus C18 (50?×?2.1?mm², 1.8?µm) for UPLC method were used. A gradient mobile phase of 20?mM anhydrous KH₂PO₄ buffer solution (containing 0.2% triethylamine (TEA), v/v) with pH adjusted to 2.9 using orthophosphoric acid (solution A) and acetonitrile (solution B) mixed in different proportions for HPLC and UPLC methods was prepared. Flow rate was set to 1.0 and 0.3?mL min^?1 for HPLC and UPLC methods, respectively, and the detection was performed for both methods at 210?nm. It worth noting that the proposed UPLC-DAD assay exhibited relatively much more precision, sensitivity, specificity, and economic and chromatographic separation superiority than proposed HPLC-UV assay. Both developed methods were compared with reference methods to prove its applicability and are suitable for purity assessment of ASP and CST in bulk and capsules.     

Analysis of Zoledronic Acid and Its Related Substances by Ion-Pair RP-LC

A simple high-performance liquid chromatographic method – ion-pair reversed- phase high performance liquid chromatography (RPIC) has been developed and employed for the analysis of zoledronic acid and its related substances in bulk material and commercial dosage forms. The mobile phase was a mixture of methanol (20%) and 5 mmol L^–1 phosphate buffer (80%) containing 6 mmol L^–1 tetrabutylammonium bromide, adjusted to pH 7.0 with sodium hydroxide. C₈ column was used as the stationary phase. The chromatographic conditions were optimized. The active ingredient – zoledronic acid was successfully separated from its related substances, including remained imidazol-1-yiacetic acid in the synthesis of zoledronic acid and other possible impurities of oxidation and decomposition. The excipients did not interfere with the determination of zoledronic acid in commercial dosage formulations. The method was rapid, simple, accurate and reproducible. It was not only successfully employed for the assay of zoledronic acid in bulk material and pharmaceutical dosage forms but also for the determination of its related substances.     

Quality-by-design-based development and validation of a stability-indicating UPLC method for quantification of teriflunomide in the presence of degradation products and its application to in-vitro dissolution

A systematic design-of-experiments was performed by applying quality-by-design concepts to determine design space for rapid quantification of teriflunomide by the ultraperformance liquid chromatography (UPLC) method in the presence of degradation products. Response surface and central composite quadratic were used for statistical evaluation of experimental data using a Design-Expert software. The response variables such as resolution, retention time, and peak tailing were analyzed statistically for the screening of suitable chromatographic conditions. During this process, various plots such as perturbation, contour, 3D, and design space were studied. The method was developed through UPLC BEH C18 2.1?×?100?mm, 1.7-µ column, mobile phase comprised of buffer (5?mM K₂HPO₄ containing 0.1% triethylamine, pH 6.8), and acetonitrile (40:60 v/v), the flow rate of 0.5?mL?min^?1 and UV detection at 250?nm. The method was developed with a short run time of 1?min. Forced degradation studies revealed that the method was stability-indicating, suitable for both assay and in-vitro dissolution of a drug product. The method was found to be linear in the range of 28–84?µg?mL^?1, 2.8–22.7?µg?mL^?1 with a correlation coefficient of 0.9999 and 1.000 for assay and dissolution, respectively. The recovery values were found in the range of 100.1–101.7%. The method was validated according to ICH guidelines.     

Method development and validation study for quantitative determination of nifedipine and related substances by ultra‐high‐performance liquid chromatography

A novel stability‐indicating reversed phase ultra‐high performance liquid chromatography (UPLC) coupled photodiode array gradient method was developed for determination of the nifedipine and related compounds. Furthermore, based on the chromatographic conditions and forced degradation studies performed through the development of the related substances method a UPLC isocratic method was validated for the determination of the assay of this active substance. An Acquity Shield RP₁₈ (50 × 3.0 mm 1.7 µm) column was used for separation of nifedipine and its five potential impurities within 11 min, which is 5‐fold less than the official method. A mobile phase consisting of 10 mm ammonium formate (pH 4.5) and methanol, delivered at a flow rate 0.5 mL/min, was employed to achieve a minimum resolution of 2.0 for all consecutive pairs of compounds. The precision value expressed as percentage relative standard deviation for method repeatability and reproducibility was <5.0%. The recoveries for all the related compounds were in the range of 99–105.0%. Linearity was found to be acceptable over the concentration range of 0.25–1.5 µg/mL for nifedipine and its impurities. The limit of quantification for nifedipine was 0.05 µg/mL, which is much less than the European Pharmacopoeia method. Copyright © 2014 John Wiley & Sons, Ltd.     

Comparison of a novel ultra-performance liquid chromatographic method for determination of retinol and α-tocopherol in human serum with conventional HPLC using monolithic and particulate columns

Retinol and α-tocopherol are biologically active compounds often monitored in blood samples because of their evident importance in human metabolism. In this study a novel ultra-performance liquid chromatographic (UPLC) method used for determination of both vitamins in human serum has been compared with conventional HPLC with particulate and monolithic C₁₈ columns. In UPLC a sub-two-micron particle-hybrid C₁₈ stationary phase was used for separation, in contrast with a five-micron-particle packed column and a monolithic column with a highly porous structure. Methanol, at flow rates of 0.48, 1.5, and 2.5 mL min⁻¹, respectively, was used as mobile phase for isocratic elution of the compounds in the three methods. Detection was performed at 325 nm and 290 nm, the absorption maxima of retinol and α-tocopherol, respectively. Analysis time, sensitivity, mobile-phase consumption, validation data, and cost were critically compared for these different chromatographic systems. Although cost and mobile-phase consumption seem to make UPLC the method of choice, use of the monolithic column resulted in almost the same separation and performance with a slightly shorter analysis time. These methods are alternatives and, in routine laboratory practice, more economical means of analysis of large numbers of biological samples than use of a traditional particulate column.     

Analytical QbD-based systematic bioanalytical HPLC method development for estimation of quercetin dihydrate

The current work entails development of rapid, sensitive, and inexpensive high-performance liquid chromatographic method of quercetin dihydrate using the quality by design approach. Quality target method profile was defined and critical analytical attributes (CAAs) were earmarked. Chromatographic separation was accomplished on a C₁₈ column using acetonitrile and ammonium acetate buffer (35:65) %v/v (containing 0.1% acetic acid, pH 3.5) as mobile phase at 0.7?mL/min flow rate with UV detector at 237?nm. Screening studies using fractional factorial design revealed that organic modifier, injection volume, column temperature, and buffer strength have significant influence on method CAAs, namely, peak area, retention time, and peak tailing. The critical method parameters were systematically optimized using Box–Behnken design. Response surface mapping was used along with numerical optimization and desirability function for identifying the optimal chromatographic conditions. Linearity was observed in the drug concentration ranging between 2 and 50?µg/mL. Accuracy analysis revealed mean % recovery between 93.6 and 96.2%, while precision study revealed mean % recovery between 93.7 and 96.5%. Limits of detection and quantification of the developed method were found to be 12.1 and 36.6?ng/mL. Overall, the studies construed successful development of chromatographic method of quercetin with enhanced method performance.     

Simultaneous Ion-Pair LC Analysis of Ephedrine and Pseudoephedrine in Rat Liver Microsomal Incubations: Application to In-Vitro Metabolism

(?)-Ephedrine (ephedrine, EPH) and (+)-ephedrine (pseudoephedrine, PEPH) are metabolized by the liver, but the species of hepatocyte cytochrome P450 (CYP450) responsible is not yet clear. To investigate which subtype of CYP450 is involved in the metabolism of EPH and PEPH, a rapid and reliable reversed-phase ion-pair liquid chromatographic method for simultaneous analysis of EPH and PEPH in rat liver microsomes has been established and validated. Matrine was selected as a suitable internal standard (IS) for calibration. After liquid?Cliquid extraction of liver microsomal samples with methyl tert-butyl ether, EPH and PEPH were separated on a C₁₈ reversed-phase column (200 mm × 4.6 mm, 5 ??m) with methanol?C0.5% sodium dodecyl sulfate?Cphosphoric acid?Ctriethylamine 60:40:1.25:1 (v/v) as mobile phase at a flow rate of 1.0 mL min. Detection was by UV absorbance at 210.5 nm. For both EPH and PEPH, calibration curves were linear over the range 1.5?C60.0 ??g mL^?1, the limit of quantification was 1.5 ??g mL^?1, and intra-day and inter-day variability was <10.0%. Average extraction recovery of the two analytes was >73%. The validated method was successfully used to study the in-vitro metabolism of EPH and PEPH. In rat liver microsomes induced by dexamethasone, enzyme activity in the metabolism of EPH and PEPH was higher than that for metabolism of phenobarbital and ??-naphthoflavone.     

Using dispersive liquid-liquid microextraction and liquid chromatography for determination of guaifenesin enantiomers in human urine

A simple, rapid, and efficient method, dispersive liquid–liquid microextraction (DLLME) coupled with high‐performance liquid chromatography‐fluorescence detector, has been developed for the determination of guaifenesin (GUA) enantiomers in human urine samples after an oral dose administration of its syrup formulation. Urine samples were collected during the time intervals 0–2, 2–4, and 4–6 h and concentration and ratio of two enantiomers was determined. The ratio of R‐(?) to S‐(+) enantiomer concentrations in urine showed an increase with time, with R/S ratios of 0.66 at 2 h and 2.23 at 6 h. For microextraction process, a mixture of extraction solvent (dichloromethane, 100 μL) and dispersive solvent (THF, 1 mL) was rapidly injected into 5.0 mL diluted urine sample for the formation of cloudy solution and extraction of enantiomers into the fine droplets of CH₂Cl₂. After optimization of HPLC enantioselective conditions, some important parameters, such as the kind and volume of extraction and dispersive solvents, extraction time, temperature, pH, and salt effect were optimized for dispersive liquid–liquid microextraction process. Under the optimum extraction condition, the method yields a linear calibration curve in the concentration range from 10 to 2000 ng/mL for target analytes. LOD was 3.00 ng/mL for both of the enantiomers.     

Direct chiral separation of caderofloxacin enantiomers by HPLC using a glycoprotein column

The enantiomers of caderoflxacin (CS-940), the new antibacterial fluoroquinolone compound, were separated on the commercially available α-acid glycoprotein-coated chiral stationary phase (Chiral-AGP) using the mobile phase of IPA: 0.15 M NaH₂PO₄ + Et₃N (pH 7.9) = 3: 97 at 0.8 mL/min with UV detection at 282 nm. The chromatographic behavior of caderofloxacin enantiomers was investigated by varying the mobile phase conditions. The chiral assay method was validated and used to determination of (R)-Caderofloxacin in (S)-Caderofloxacin raw material samples.     

Separation and determination of six catechins in tea by pressurized capillary electrochromatography

An efficient pressurized capillary electrochromatography (pCEC) method has been successfully developed for the determination of six catechins in tea. The separation was performed on a reversed-phase EP-100-20/45-3-C18 capillary column (total length of 45?cm, effective length of 20?cm, diameter of 100?μm, ODS packing inside for 3?μm). The mobile phase ratio of organic phase, the concentration of phosphate buffer and sodium heptanesulfonate, separation voltage, and other experimental conditions were investigated and optimized. The mobile phase was 15?mM NaH₂PO₄ and 12?mM sodium heptanesulfonate (pH 3.0)/methanol (64:36) at a flow rate of 0.04?mL/min. Under optimal conditions including applied voltage of ?4?kV and a UV detection wavelength of 230?nm, the six catechins in the tea were well separated. The calibration curves for the analytes had good linearity in the range of 8.02?μg/mL–202.13?μg/mL with a correlation coefficient of 0.9928–0.9997. The limits of detection (LOD) for the six catechins were 4.62?μg/mL–11.63?μg/mL (S/N?=?3). The recoveries of the six catechins were 96.2%–108.4% with a relative standard deviation (RSD) between 0.78% and 4.51%. The method has been used for the determination of six catechins in tea samples with good results.     

高效液相色谱法同时测定尿液中4种非蛋白氮的含量

建立了高效液相色谱(HPLC)同时测定尿液中4种常见的非蛋白氮物质--肌酸(Cr)、尿肌酐(Cn)、尿酸(Ua)及假尿嘧啶核苷(Pu)含量的方法。尿液首先经丙酮沉淀法除去大分子化合物,然后冷冻干燥,复溶后直接进行HPLC分析。色谱分离选用Waters RP18 Column (150 mm×4.60 mm,3.5 μm),以10.0 mmol/L磷酸缓冲溶液 (pH 4.78)和乙腈为流动相进行梯度洗脱,流速为0.8 mL/min,于220 nm波长下检测,可在7 min内完成4种非蛋白氮的快速分离分析。结果表明Cr、Cn、Ua、Pu在0.1~250 mg/L范围内线性关系均良好(相关系数均大于0.999),检出限分别为9.31、26.19、4.70、6.30 μg/L,加标回收率为81%~111%,峰高的相对标准偏差(RSD, n=3)为0.23%~2.78%,满足定量要求。该法简便、快速,结果准确可靠,为2型糖尿病(T2DM)患者肾功能损坏的研究提供了检测手段。     

应用质量源于设计的理念优化大鼠血浆中大黄蒽醌的分析方法

应用质量源于设计理念建立一种高效液相色谱-荧光检测法(HPLC-FLD)用于测定大鼠血浆中5种大黄蒽醌。用Plackett-Burman设计考察流动相中甲醇含量、pH值、流速、柱温和进样体积对色谱峰的分离度、理论塔板数、最末洗脱峰的保留时间和拖尾因子的影响,结果显示流动相中甲醇含量、流速和柱温对色谱系统的影响显著(p<0.05)。继而采用Box-Behnken设计结合响应面法考察上述三因素对分离度、保留时间和理论塔板数的影响。用Derringer渴求函数评价了响应值的综合作用。得出最优色谱条件为:以甲醇-0.1%(v/v)磷酸水溶液(81.4:18.6, v/v)为流动相等度洗脱,流速1.1 mL/min,柱温31℃,荧光检测激发波长为440 nm,发射波长为540 nm。建立的模型显示良好的预测性。结果表明:质量源于设计的理念可有效地应用于优化高效液相色谱分析方法。     

Determination of Amphotericin-B in Human Plasma by Reversed-Phase High Performance Liquid Chromatography Using a Short Octyl Column

Abstract

Amphotericin-B is a polyene antifungal antibiotic used for the treatment of severe systemic fungal infections. For effective treatment of urinary fungaria and the prevention of significant adverse-effects, monitoring the concentration of Amphotericin-B in biological samples of humans (ingesting the drug) is required. In this experiment, Amphotericin-B was isolated from plasma endogenous substances by adding 200 μL of acetonitrile in 800 μL of plasma. This mixture was vortex mixed, 20 mg of zinc sulfate and 10 mg of monobasic potassium phosphate was added to the mixture. This mixture was again vortex mixed and followed by centrifugation. The supernatant was filtered through a 0.45 μm membrane and a 100 μL aliquot of this solution was injected onto the chromatographic system. A short column of 60 mm × 4.6 mm packed with 3 μm octyl particles was used with an isocratic elution of 50/50, acetonitrile/0.01M KH₂PO₄ (v/v). The pH of the mobile phase mixture was adjusted to 3.5 with H₃PO₄. The intact drug molecule (parent drug) was monitored by a W-visible detector at 410 nm and 0.10-0.005 A.U.F.S. The limits of detection of the method were 0.03 μg/mL for 100 μl injection volume at signal-to-noise ratio of 3.     

超高效液相色谱法测定湿巾中的三氯羟基二苯醚

采用超高效液相色谱法(UPLC)检测湿巾中的三氯羟基二苯醚含量.湿巾样品加甲醇超声提取,离心过滤后上机,采用甲醇-水(体积比为8∶2)为流动相,Phenomenex C18色谱柱分离,在波长280 nm下检测,外标法定量.结果表明:方法在0.50~100.00 mg/L质量浓度范围内线性良好,相关系数为0.999 1,检出限为0.30 mg/kg,方法定量限为10.0 mg/kg,RSD为2.3%,加标回收率为90.50%~93.50%.方法前处理简便快捷,准确度、精密度、稳定性好,检测结果准确可靠.     

Simultaneous Determination of Mixtures of Nitrite and Nitrate by Reversed‐Phase Ion‐Pair Liquid Chromatography

To overcome the relatively low resolution in the separation and quantitative analysis of mixtures of nitrite and nitrate, a reversed‐phase ion‐pair liquid chromatographic method is developed with advantages of high accuracy, good selectivity, high efficiency, and low cost. By employing cetyltrimethylammonium bromide (CTAB) as the ion‐pair reagent, this method can work excellently in situations where one component in the mixture is highly in excess (e.g., molar ratio of n( NO₂⁻):n(NO₃⁻) ranging from 1:99 to 95:5). The operation parameters were optimized on a Shim‐pack VP‐ODS(150 L × 4.6) analytical column using a methanol/water ratio of 50:50 (v/v) mobile phase containing 7.0 mmol/L CTAB and 3.1 mmol/L potassium dihydrogen phosphate (KH₂PO₄).The column works at a temperature of 35 °C with a flow rate of 1.5 mL/min. Such a protocol can be applied to monitor the formation of trace nitrous acid during the oxidative decomposition of nitric acid.     

A Chemometrical Approach to Optimization and Validation of an HPLC Assay for Rizatriptan and its Impurities in Tablets

Abstract

An isocratic reversed‐phase high‐performance liquid chromatographic method was developed and validated for the analysis of a novel antimigraine drug, rizatriptan benzoate, in a dosage form along with its two impurities, L‐749.019 and L‐783.540. The method used a C₁₈ XTerra? (150×3.9 mm), 5 µm column. The mobile phase consisted of a mixture of methanol, TEA (1%) and 10 mM KH₂PO₄ (5:9.5:85.5 v/v) at a flow rate of 1.2 ml min^?1 (pH of the water phase was adjusted to 5.5 with 85% orthophosphoric acid). Column temperature was 20°C and the detection was performed at 225 nm. The central composite design technique and the response surface method were used in the robustness test considerations. The method was applied satisfactorily to the analysis of commercial rizatriptan formulation.     

A Reverse Phase HPLC Method to Determine Six Food Dyes Using Buffered Mobile Phase.

Abstract

A reverse phase high performance liquid chromatographic (HPLC) method to determine six food dyes (Sunset Yellow (E-110), Carminic acid (E-120) Carmoisine (E-122), Amaranth (E-123), Ponceau 4R (E-124) and Erythrosine (E-127) is developed in this paper. The separation was made on a Nova-Pack C18 column using methanol -NaH₂PO₄/Na₂HPO₄ pH=7 buffer solution 0.1M as mobile phase with an elution gradient system. The detection was made with a variable UV-Vis. detector fixed at 520 nm.

The effect of mobile phase composition such as the percentage of methanol or acetonitrile, pH value and ionic strength on retention times of the dyes was investigated. In the chromatographic conditions selected, the dyes were eluted in four minutes. Two calibration graphs for each dye were established by measuring the peak area and the peak height in the chromatograms. Determination limits ranging from 0.8 to 9.2 ng were obtained when the peak area was measured.

Several commercial products containing some of these dyes were analyzed.     

Analysis of free amino acids in Amur sturgeon by ultra‐performance liquid chromatography using pre‐column derivatization with 6‐aminoquinolyl‐carbamyl

A rapid, sensitive, and reliable ultra‐performance liquid chromatography (UPLC) coupled with photodiode array detection method was developed for the amino acid analysis of Amur sturgeon (Acipenser schrenckii Brandt). The method uses minimal sample volume and automated online precolumn derivitization of amino acids with fluorescent 6‐aminoquinolyl‐carbamyl reagent. The chromatographic separation was achieved by UPLC, which used a column with 1.7 μm particle packing that enabled higher speed of analysis, peak capacity, greater resolution, and increased sensitivity. Amino acid derivatives obtained under optimal conditions were separated on a Waters UPLC BEH C₁₈ column with Acetonitrile–acetate buffer as mobile phase. Matrix effects were investigated and good linearities with correlation coefficients better than 0.9949 were obtained over a wide range of 5–1000 μmol/L for all amino acids. The simple sample preparation and minimal sample volume make the method useful for the quantitation of 17 amino acids in Amur sturgeon samples. It is concluded that a rapid and robust platform based on UPLC was established, and a total of 17 amino acids of Amur sturgeon were tentatively detected. This method showed good accuracy and repeatability that can be used for the quantification of amino acids in real samples.