Determination of fatty acids by high-performance liquid chromatography and fluorescence detection using precolumn derivatization with 9-fluorenylmethyl chloroformate

A new high-performance liquid chromatographic method is described for the determination of fatty acids in seed oils. The method was based on precolumn derivatization with 9-fluorenylmethyl chloroformate as a labeling agent and fluorescence detection. Fatty acids were extracted from the samples and subjected to derivatization with the reagent at 60°C for 10?min. The chromatographic separation of 14 fatty acids (C10–C22) was achieved on a combined loading compression octadecyl sulfate (CLC-ODS) column with a run time of 30?min. Three-step gradient elution of a mobile phase consisted of acetonitrile and water was used, and the signal was monitored at excitation and emission wavelengths of 265 and 315?nm, respectively. The method indicated favorable sensitivity and reproducibility for fatty acids’ derivatives. The detection limits, at a signal-to-noise ratio of 3, were 0.01–0.05?µg/ml and relative standard deviations (RSDs) were less than 0.27%. Excellent linear responses were observed with coefficients of 0.9995. This method was applied to quantify fatty acids in white, brown, and black sesame seeds’ oil.     

LC–UV Methodology for Simultaneous Determination of Lamivudine and Zidovudine in Plasma by Liquid–Liquid Extraction

This study describes an accurate, sensitive, and specific chromatographic method for the simultaneous quantitative determination of lamivudine and zidovudine in human blood plasma, using stavudine as an internal standard. The chromatographic separation was performed using a C8 column (150 × 4.6 mm, 5 μm), and ultraviolet absorbency detection at 270 nm with gradient elution. Two mobile phases were used. Phase A contained 10 mM potassium phosphate and 3% acetonitrile, whereas Phase B contained methanol. A linear gradient was used with a variability of A-B phase proportion from 98–2% to 72–28%, respectively. The drug extraction was performed with two 4 mL aliquots of ethyl acetate.     

LC Determination of Nosiheptide in Swine Kidney and Liver

A liquid chromatographic method was developed for determination of nosiheptide in swine kidney and liver. The tissue samples were extracted with acetonitrile and defatted with hexane. The analytes were determined at a fluorescence excitation/emission wavelength of 357/515 nm and with gradient elution program. The limits of detection and limits of quantification were 5 and 20 ng g^?1, respectively, for swine kidney and liver. The mean recoveries for nosiheptide in swine kidney and liver ranged from 70.3 to 97.4% with coefficients of variation below 11.3% at 20–100 ng g^?1 fortification levels.     

Simple, Rapid, and Accurate RP-HPLC Method for Determination of Cystine in Human Urine after Derivatization with Dansyl Chloride

An accurate, simple, and sensitive reversed-phase high-performance liquid chromatographic method, with loratadine as internal standard (IS) and UV detection at 286 nm, has been developed for deterination of cystine in human urine. The major innovations of the method include use of acrylonitrile to protect cysteine from oxidization to cystine, separation of cysteine, as the dansyl derivative, from cystine, and use of isocratic elution instead of gradient elution to reduce the time and cost of serial analysis. The mobile phase was 0.05 M sodium acetate–methanol, 35:65 (v/v), adjusted to pH 3.5 with 2.5 M citric acid, at a flow rate of 1.0 mL min^?1. The retention times of cystine and the IS were 16.6 and 19.9 min, respectively. The limit of detection for cystine was 0.3 mg L^?1. Extraction recovery of cystine was >85.6%. Intra-day and inter-day precision (RSD) for cystine were below 4.3 and 8.5%, respectively. There was no chromatographic interference from other α-amino acids present in mammalian proteins, or from other urine components. The calibration plot for the cystine derivative was linear in the range 1–500 mg L^?1 and the correlation coefficient was 0.9992. The method was validated appropriately and successfully used for determination of cystine in human urine.     

LC–UV Methodology for Simultaneous Determination of Lamivudine and Zidovudine in Plasma by Liquid–Liquid Extraction

This study describes an accurate, sensitive, and specific chromatographic method for the simultaneous quantitative determination of lamivudine and zidovudine in human blood plasma, using stavudine as an internal standard. The chromatographic separation was performed using a C8 column (150 × 4.6 mm, 5 μm), and ultraviolet absorbency detection at 270 nm with gradient elution. Two mobile phases were used. Phase A contained 10 mM potassium phosphate and 3% acetonitrile, whereas Phase B contained methanol. A linear gradient was used with a variability of A-B phase proportion from 98–2% to 72–28%, respectively. The drug extraction was performed with two 4 mL aliquots of ethyl acetate.

     

LC–MS–MS Analysis of Strictosamide in Rat Plasma, and Application of the Method to a Pharmacokinetic Study

A rapid and simple high-performance liquid chromatographic tandem mass spectrometric method has been developed and validated for analysis of strictosamide in rat plasma. Chromatographic separation was achieved on a C₁₈ column by gradient elution with mixtures of methanol, water, and acetonitrile containing 0.05% acetic acid. Digoxin was used as internal standard. Selected reaction monitoring (SRM) was used for MS quantitation. Linearity was good in the range 0.05–20 ng mL^?1 in rat plasma. The lower limit of quantitation was 0.04 ng mL^?1. The method is precise and reliable and can be applied to pharmacokinetic studies.     

Establishing the chromatographic fingerprint of traditional Chinese medicine standard decoction based on quality by design approach: A case study of Licorice

A novel analytical quality by design approach for developing a chromatographic fingerprint was established for analyzing complex traditional Chinese medicine, using a licorice standard decoction as an example. Considering the characteristics of integrity and ambiguity, the resolution of eight common peaks, total peak number, capacity factor distributions, and peak purity were selected as potential critical method attributes for assessing the quality of the chromatographic fingerprint. A central composite design was used to evaluate the relationship between critical method attributes and critical method parameters, including column temperature, wavelength, flow rate, formic‐acid concentration, and gradient parameters. A standard probability method was employed to calculate the design space of the fingerprint analysis parameters and evaluate the robustness of the methodology. The optimized high‐performance liquid chromatography fingerprint conditions were acetonitrile and 0.1% formic acid water gradient elution (0‐5 min, 5–19% A; 5–10 min, 19% A; 10–50 min, 19–42% A; 50–54 min, 42–100% A; 54–60 min, 100% A), column temperature 25±5°C, detection wavelength 265 nm. The design space of fingerprint analytical method based on the analytical quality by design approach not only met the requirements of the fingerprint analysis, but also improved the robustness and applicability of the fingerprint method.     

Fingerprint analysis and multi‐ingredient quantitative analysis for quality evaluation of Xiaoyanlidan tablets by ultra high performance liquid chromatography with diode array detection

A rapid and sensitive ultra high performance liquid chromatography method with diode array detection was developed for the fingerprint analysis and simultaneous determination of seven active compounds in Xiaoyanlidan (XYLD) tablets. The chromatographic separations were obtained on an Agilent Eclipse plus C₁₈ column (50 × 2.1 mm id, 1.8 μm) using gradient elution with water/formic acid (1%) and acetonitrile at a flow rate of 0.4 mL/min. Within 63 min, 36 peaks could be selected as the common peaks for fingerprint analysis to evaluate the similarities among several samples of XYLD tablets collected from different manufacturers. In quantitative analysis, seven compounds showed good regression (R > 0.9990) within test ranges and the recovery of the method was within the range of 95.9–104.3%. The method was successfully applied to the simultaneous determination of seven compounds in six batches of XYLD tablets. These results demonstrate that the combination of chromatographic fingerprint analysis and simultaneous multi‐ingredient quantification using the ultra high performance liquid chromatography method with diode array detection offers a rapid, efficient, and reliable approach for quality evaluation of XYLD tablets.     

Simultaneous quantification of the major bile acids in Artificial Calculus bovis by high‐performance liquid chromatography with precolumn derivatization and its application in quality control

An accurate and sensitive high‐performance liquid chromatography method coupled with ultralviolet detection and precolumn derivatization was developed for the simultaneous quantification of the major bile acids in Artificial Calculus bovis, including cholic acid, hyodeoxycholic acid, chenodeoxycholic acid, and deoxycholic acid. The extraction, derivatization, chromatographic separation, and detection parameters were fully optimized. The samples were extracted with methanol by ultrasonic extraction. Then, 2‐bromine‐4’‐nitroacetophenone and 18‐crown ether‐6 were used for derivatization. The chromatographic separation was performed on an Agilent SB‐C18 column (250 × 4.6 mm id, 5 μm) at a column temperature of 30°C and liquid flow rate of 1.0 mL/min using water and methanol as the mobile phase with a gradient elution. The detection wavelength was 263 nm. The method was extensively validated by evaluating the linearity (r² ≥ 0.9980), recovery (94.24–98.91%), limits of detection (0.25–0.31 ng) and limits of quantification (0.83–1.02 ng). Seventeen samples were analyzed using the developed and validated method. Then, the amounts of bile acids were analyzed by hierarchical agglomerative clustering analysis and principal component analysis. The results of the chemometric analysis showed that the contents of these compounds reflect the intrinsic quality of artificial Calculus bovis, and two compounds (hyodeoxycholic acid and chenodeoxycholic acid) were the most important markers for quality evaluating.     

Determination of Biogenic Amines in Digesta by High Performance Liquid Chromatography with Precolumn Dansylation

A rapid high-performance liquid chromatographic method for the determination of nine biogenic amines in digesta sample from pig cecum and colon was developed using a simple direct derivatization with dansyl chloride. After precipitation with trichloroacetic acid and extraction by n-hexane, the amines were separated on a C₁₈ column using a water–acetonitrile gradient elution program. The calibration curve for each amine was linear over the range of 50 to 250 µmol/L, and the relative standard deviation for intra-assay precision was below 0.5%. The recovery ranged from 86.79% to 117.62% while the limit of detection was 0.1 µmol/L for the amines except for spermine with a value of 0.03 µmol/L. This procedure was successfully applied to identify and quantify biogenic amines in the hindgut digesta of the pig.     

Stress Degradation Behavior of Desipramine Hydrochloride and Development of Suitable Stability-Indicating LC Method

A simple reverse phase liquid chromatographic method was developed for the quantitative determination of desipramine hydrochloride and its related impurities in bulk drugs which is also stability-indicating. During the forced degradation at hydrolysis, oxidative, photolytic and thermal stressed conditions, the degradation results were only observed in the oxidative stress condition. The blend of the degradation product and potential impurities were used to optimize the method by an YMC Pack Pro C₁₈ stationary phase. The LC method employs a linear gradient elution with the water–acetonitrile–trifluoroacetic acid as mobile phase. The flow rate was 1.0 mL min^?1 and the detection wavelength 215 nm. The stressed samples were quantified against a qualified reference standard and the mass balance was found close to 99.0% (w/w) when the response of the degradant was considered to be equal to the analyte (i.e. desipramine). The developed RP-LC method was validated in agreement with ICH requirements.     

Comparison between core–shell and totally porous particle stationary phases for fast and green LC determination of five hepatitis‐C antiviral drugs

The performances of core–shell 2.7 μm and fully porous sub‐2 μm particles packed in narrow diameter columns were compared under the same chromatographic conditions. The stationary phases were compared for fast separation and determination of five new antiviral drugs; daclatasvir, sofosbuvir, velpatasvir, simeprevir, and ledipasvir. The gradient elution was done using ethanol as green organic modifier, which is more environmentally friendly. Although both columns provided very good resolution of the five drugs, core–shell particles had proven to be of better efficiency. Under gradient elution conditions, core–shell particles exhibited faster elution, better peak shape, and enhanced resolution adding to lower system backpressure. The column backpressure on sub‐2 μm particles was more than twice that on core–shell particles. This gives a chance to use conventional high‐performance liquid chromatography conditions without needing special instrumentation as that required for ultra‐high performance liquid chromatography. The method was validated for determination of the five drugs by gradient elution using mobile phase composed of organic modifier ethanol and aqueous part containing 0.75 g sodium octane sufonate and 3.0 g sodium dihydrogen phosphate per liter at pH of 6.15. Detection was done using UV‐detector set at 210 nm. The linearity, accuracy, and precision were found very good within the concentration range of 2–200 μg/mL.     

Determination of Armillarisin A in Rat Plasma by SPE and LC-MS-MS

A rapid, sensitive and convenient ultra-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of armillarisin A in rat plasma. Following hydrophilic–lipophilic balance solid-phase extraction, armillarisin A and propylparaben (internal standard) were separated using a gradient elution program on a C₁₈ column and detected by mass spectrometry in the negative ion mode with the multiple reaction monitoring mode. The total chromatographic running time was short (1.8 min). The method was linear over the concentration range of 0.5–250.00 ng mL^?1 for armillarisin A. The lower limit of quantification of armillarisin A was 0.5 ng mL^?1, using as little as 100 μL plasma. The intra-day and inter-day relative standard deviations were less than 15% and the relative errors were all within 10%. The extraction recovery for armillarisin A was approximately 100%, and no absolute matrix effect was observed. Finally, the method was successfully applied to a preclinical pharmacokinetic study of armillarisin A in four rats following an intravenous administration.     

Development and validation of a cost-effective and sensitive bioanalytical HPLC-UV method for determination of lopinavir in rat and human plasma

A simple, sensitive and cost-effective HPLC-UV bioanalytical method for determination of lopinavir (LPV) in rat and human plasma was developed and validated. The plasma sample preparation procedure includes a combination of protein precipitation using cold acetonitrile and liquid–liquid extraction with n-hexane–ethyl acetate (7:3, v/v). A good chromatographic separation was achieved with a Phenomenex Gemini column (C₁₈, 150 mm × 2.0 mm, 5 μm) at 40°C with gradient elution, at 211 nm. Calibration curves were linear in the range 10–10,000 ng/mL, with a lower limit of quantification of 10 ng/mL using 100 μL of plasma. The accuracy and precision in all validation experiments were within the criteria range set by the guidelines of the Food and Drug Administration. This method was successfully applied to a preliminary pharmacokinetic study in rats following an intravenous bolus administration of LPV. Moreover, the method was subsequently fully validated for human plasma, allowing its use in therapeutic drug monitoring (TDM). In conclusion, this novel, simple and cost-efficient bioanalytical method for determination of LPV is useful for pharmacokinetic and drug delivery studies in rats, as well as TDM in human patients.     

Simultaneous determination of thirteen main components and identification of eight major metabolites in Xuebijing Injection by UPLC/Q-TOF

An ultra performance liquid chromatographic method was used for the simultaneous identification and quantification of thirteen main components in Xuebijing Injection, including uridine, gallic acid, guanosine, danshensu, protocatechualdehyde, oxypaeoniflorin, hydroxysafflor yellow A, paeoniflorin, ferulic acid, safflor yellow A, senkyunolide I, senkyunolide H and salvianolic acid B. The chromatographic separation was performed on an Acquity UPLC BEH C₁₈ column (1.7-μm, 2.1 × 100 mm, i.d.) with a gradient elution of acetonitrile and 0.2% acetic acid at a flow rate of 0.4 mL/min. The method was validated for linearity (r ² > 0.9990), intra- and inter-day precision (RSD < 1.94%), accuracy (91.8–99.7%), recovery (96.8–103.8%), limits of detection (0.16–8.0 ng), and limits of quantification (0.54–26.8 ng). At least eight metabolites in prototype were found in rat plasma and urine after intravenous injection of 4 mL/kg doses of Xuebijing Injection. The proposed method could be utilized to qualify and control Xuebijing Injection to ensure its safety and efficacy in application.     

LC–DAD and LC–ESI–MS Chromatographic Fingerprinting and Quantitative Analysis for Evaluation of the Quality of Huang-Lian-Jie-Du-Tang

LC–DAD coupled with electrospray tandem mass spectrometry (LC–ESI–MS–MS) has been used to evaluate the quality of the traditional Chinese medicine Huang-Lian-Jie-Du-Tang (HLJDT). Twenty-five chromatographic peaks were obtained from a C₁₈ analytical column by gradient elution with acetonitrile and formate buffer (containing 0.5% formic acid) at a flow rate of 1.0 mL min^?1. The linearity, precision, and accuracy of the data obtained were acceptable. Thirteen components were identified by ESI–MS, and seven of these were quantitatively analyzed by LC–DAD. The method was used to analyze ten batches of HLJDT, and both chromatographic fingerprints and quantitative data were used to evaluate the quality of the HLJDT. It was concluded that this LC–DAD–ESI–MS method enables more fully validated and complete evaluation and monitoring of the quality of HLJDT.     

Highly sensitive HPLC‐DAD method for the assay of gefitinib in patient plasma and cerebrospinal fluid: application to a blood‐brain barrier penetration study

The quantification of intracranial gefitinib (GEF) exposure is limited owing to the sensitivity of analytical equipment. Although mass spectrometry (MS) is the preferred method because of its high sensitivity, the equipment is not available in many laboratories, especially in developing Asian countries. In this paper, we developed a highly sensitive high performance liquid chromatography‐diode array detector (HPLC‐DAD) method for the assay of GEF in human cerebrospinal fluid (CSF) and plasma. GEF was extracted from CSF and plasma by solid‐phase extraction and liquid–liquid extraction, respectively. The chromatographic separation was performed on a C₁₈ column with gradient elution of 0.1% triethylamine solution and acetonitrile, then finally detected at 344 nm. This method was validated and proved to be highly sensitive with a lower limit of quantitation value of 0.11 ng/mL in CSF and 11 ng/mL in plasma. The blood–brain barrier penetration ratio of GEF ranged from 1.48 to 2.41%. This method provides a reliable MS‐independent solution for the quantitation of GEF in patients’ CSF and plasma. Copyright © 2015 John Wiley & Sons, Ltd.     

Stress Degradation Behavior of Desipramine Hydrochloride and Development of Suitable Stability-Indicating LC Method

A simple reverse phase liquid chromatographic method was developed for the quantitative determination of desipramine hydrochloride and its related impurities in bulk drugs which is also stability-indicating. During the forced degradation at hydrolysis, oxidative, photolytic and thermal stressed conditions, the degradation results were only observed in the oxidative stress condition. The blend of the degradation product and potential impurities were used to optimize the method by an YMC Pack Pro C₁₈ stationary phase. The LC method employs a linear gradient elution with the water–acetonitrile–trifluoroacetic acid as mobile phase. The flow rate was 1.0 mL min⁻¹ and the detection wavelength 215 nm. The stressed samples were quantified against a qualified reference standard and the mass balance was found close to 99.0% (w/w) when the response of the degradant was considered to be equal to the analyte (i.e. desipramine). The developed RP-LC method was validated in agreement with ICH requirements.

     

反相硅胶整体柱离子对色谱法快速测定吡啶离子液体阳离子

建立了整体柱离子对色谱-紫外检测法梯度淋洗快速分离测定4种吡啶离子液体阳离子的方法。分离采用C18反相硅胶整体柱,以离子对试剂(用柠檬酸调节pH值)-乙腈为淋洗液,并采用多级梯度洗脱程序。实验考察了色谱柱、离子对试剂、乙腈浓度、色谱柱温度及流速对吡啶阳离子保留的影响,并讨论了其保留规律。咪唑阳离子的保留符合碳数规律。最佳色谱条件是:在流速3.0 mL/min,柱温30℃下,以1.0 mmol/L庚烷磺酸钠(pH 4.0)(A)+乙腈(B)为淋洗液进行梯度洗脱。淋洗梯度为0～2.0 min,10%B;2.0～2.5 min,10%～15%B;2.5～4.0 min,15%B;4.0～4.5 min,15%～20%B;4.5～10.0 min,20%B。在此条件下,4种吡啶阳离子可在7 min内基线分离。所测阳离子的检出限(S/N=3)为0.05～0.17 mg/L;峰面积的相对标准偏差(n=5)小于0.6%。将本方法用于实验室合成的离子液体样品和污水样品的分析,加标回收率在95.7%～99.0%之间。本方法准确、快速,具有较好的实用性。