Waters Acquity Ultra-performance Liquid Chromatography Research Articles

Simultaneous Determination of Eight Bioactive Constituents in Shensong Yangxin Capsule by UPLC

ObjectiveTo develop a ultra performance liquid chromatography (UPLC) method for the quality evaluation of Shensong Yangxin Capsule (SYC). MethodsThe Waters Acquity UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm) was used. Acetonitrile and water containing 0.1% phosphoric acid were used as mobile phases of UPLC with gradient elution. The detection wavelengths were set at 203 (ginsenoside Rb1), 286 (salvianolic acid B), 230 (paeoniflorin), 221 (schisantherin A), 280 (sodium danshensu), 327 (chlorognenic acid), 335 (spinosin), and 345 nm (berberine hydrochloride), respectively. The flow rate was set at 0.4 mL/min and column temperature was 35 °C. ResultsThe contents of paeoniflorin, salvianolic acid B, schisantherin A, sodium danshensu, chlorognenic acid, spinosin, berberine hydrochloride, and ginsenoside Rb1 were determined from 10 batches of SYC. ConclusionThe method of the quality evaluation of SYC has acceptable precision, reproducibility, and stability, which could be used as a new method for the quality control of SYC.

Fast Determination of Adenosine 5′-Triphosphate (ATP) and Its Catabolites in Royal Jelly Using Ultraperformance Liquid Chromatography

To obtain insight into the metabolic regulation of adenosine 5'-triphosphate (ATP) in royal jelly and to determine whether ATP and its catabolites can be used as objective parameters to evaluate the freshness and quality of royal jelly (RJ), a rapid ultraperformance liquid chromatography (UPLC) method has been developed for feasible separation and quantitation of ATP and its catabolites in RJ, namely, adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), inosine monophosphate (IMP), inosine (HxR), and hypoxanthine (Hx). The analytes in the sample were extracted using 5% precooled perchloric acid. Chromatographic separation was performed on a Waters Acquity UPLC system with a Waters BEH Shield RP18 column and gradient elution based on a mixture of two solvents: solvent A, 50 mM phosphate buffer (pH 6.5); and solvent B, acetonitrile. The recoveries were in the range of 86.0-102.3% with RSD of no more than 3.6%. The correlation coefficients of six analytes were high (r(2) ≥ 0.9988) and within the test ranges. The limits of detection and quantification for the investigated compounds were lower, at 0.36-0.68 and 1.22-2.30 mg/kg, respectively. The overall intra- and interday RSDs were no more than 1.8%. The developed method was successfully applied to the analysis of the analytes in samples. The results showed that ATP in RJ sequentially degrades to ADP, AMP, IMP, HxR, and Hx during storage.

Simultaneous Determination of 25 Common Pharmaceuticals in Whole Blood Using Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry

An ultra-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of 25 common pharmaceuticals in whole blood. The selected pharmaceuticals represent the most frequently detected drugs in our forensic laboratory with basic properties such as analgesics, antidepressants, antihistamines, antihypertensives, antipsychotics and β-blockers. Whole blood samples were extracted with butyl acetate after adjusting pH with 2M NaOH. The target analytes were separated on a 100 × 2.1 mm ACQUITY BEH 1.7 µm C18 column by a formic acid/acetonitrile gradient elution using a Waters ACQUITY Ultra-Performance Liquid Chromatography system. Quantification was performed on a Waters tandem quadrupole ACQUITY TQD using multiple reaction monitoring in positive mode. The analytes were eluted within 11 min. The limit of quantification (LOQ) ranged from 0.002 to 0.01 mg/kg depending on the analyte. A good linear behavior was achieved for all analytes in the range from LOQ to 1.0 or 2.0 mg/kg blood. The absolute recoveries were between 55-87% for all compounds except norfluoxetine (44%). The method showed acceptable precision and accuracy for almost all analytes. Only unstable compounds like levomepromazine, methylphenidate, mirtazapine, norfluoxetine and zuclopenthixol deviated more. The method was successfully applied to more than 200 authentic blood samples within a year from forensic investigations.

Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Bupropion and its Main Metabolites in Human Whole Blood

A selective and sensitive ultra-performance liquid chromatography (UPLC)-electrospray ionization-tandem mass spectrometry (MS) method for simultaneous determination of bupropion and its main metabolites, hydroxybupropion, erythrohydrobupropion, and threohydrobupropion, in human whole blood is presented. The sample preparation consists of cleanup protein precipitation with methanol combined with a solid-phase extraction on Oasis HLB cartridges. Analytes were separated on a Waters Acquity UPLC((R)) BEH phenyl column using a binary mobile phase consisting of ammonium formate buffer (2 mM, pH 4) and acetonitrile. Detection was performed on a Waters Acquity UPLC system coupled to a Quattro Premier triple-quadrupole MS in positive ion selected reaction monitoring. Internal standards were bupropion-d(9) and hydroxybupropion-d(6). Linearity was from 5 to 1000 ng/mL for bupropion and from 10 to 2000 ng/mL for metabolites. Accuracy profiles (80-120%), precision (< 15%), and limits of detection (1 ng/mL for bupropion and 2 ng/mL for metabolites) were also evaluated and responded to all criteria of validation. The aim of this study was to compare this presented method with a previously described method developed on a classic liquid chromatography-tandem MS system.

Separation of microcystins and nodularins by ultra performance liquid chromatography

Four ultra performance liquid chromatography (UPLC) columns with different reversed-phase characteristics were tested in the chromatographic separation of 10 microcystins and three nodularins, cyanobacterial peptide toxins. The columns had been designed by the manufacturer to withstand the ultra-high pressure generated by sub-2 μm stationary phase particles and the Waters ACQUITY UPLC system in ultra-fast separations. The gradient mobile phase consisted of water and acetonitrile, both acidified with trifluoroacetic acid, with three gradient rise times: 1, 1.5 and 2 min. The UV detection of the toxins was performed by a photodiode array detector. The chromatographic performance was evaluated both visually and by calculating chromatographic parameters such as capacity factor, resolution, peak width at half height, selectivity and peak asymmetry. The best chromatographic performance as judged by visual inspection was given by the ACQUITY BEH Shield RP18 and ACQUITY BEH Phenyl columns. The BEH Shield RP18 column showed excellent selectivity and resolution of chosen peak pairs considered as critical. A further advantage of the UPLC system was the high sample throughput with a total analysis time of 3.12 min (injection-to-injection) equalling to 461 separations per 24 h.

Quantification of 8 HIV-Protease Inhibitors and 2 Nonnucleoside Reverse Transcriptase Inhibitors by Ultra-Performance Liquid Chromatography with Diode Array Detection

Most HPLC-UV methods for therapeutic drug monitoring of anti-HIV drugs have long run times, which reduce their applicability for high-throughput analysis. We developed an ultra-performance liquid chromatography (UPLC)-diode array detection method for the simultaneous quantification of the HIV-protease inhibitors (PIs) amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, saquinavir, and tipranavir (TPV), and the nonnucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz and nevirapine. Solid-phase extraction of 1 mL plasma was performed with Waters HLB cartridges. After 3 wash steps, we eluted the drugs with methanol, evaporated the alcohol, and reconstituted the residue with 50 microL methanol. We injected a 4-microL volume into the UPLC system (Waters ACQUITY UPLC BEH C8 column maintained at 60 degrees C) and used a linear gradient of 50 mmol/L ammonium acetate and 50 mmol/L formic acid in water versus acetonitrile to achieve chromatographic separation of the drugs and internal standard (A-86093). Three wavelengths (215, 240, and 260 nm) were monitored. All drugs were eluted within 15 min. Calibration curves with concentrations of 0.025-10 mg/L (1.875-75 mg/L for TPV) showed coefficients of determination (r(2)) between 0.993 and 0.999. The lower limits of quantification were well below the trough concentrations reported in the literature. Inter- and intraassay CVs and the deviations between the nominal and measured concentrations were <15%. The method was validated by successful participation in an international interlaboratory QC program. This method allows fast and simultaneous quantification of all commercially available PIs and NNRTIs for therapeutic drug monitoring.

Simultaneous determination of 21 preservatives in cosmetics by ultra performance liquid chromatography

An ultra performance liquid chromatography (UPLC) method has been developed for the simultaneous determination of 21 preservatives: 2-methyl-4-isothiazoline-3-ketone, bronopol, 5-chloro-2-methyl-4-isothiazoline-3-ketone, benzyl alcohol, 2-phenoxyethanol, methyl-p-hydroxy benzoate, ethyl-p-hydroxy benzoate, methyl benzoate, 4-hydroxybenzoic acid iso-propyl ester, propyl-p-hydroxy benzoate, 4-chloro-3-methylphenol, ethyl benzoate, 2-phenylphenol, 4-hydroxybenzoic acid iso-butyl ester, butyl-p-hydroxy benzoate, 4-chloro-3,5-dimethylphenol, phenyl benzoate, 2,4-dichloro-3,5-dimethylphenol, 2-benzyl-4-chlorophenol, triclocarban and triclosan in cosmetics. A Waters ACQUITY UPLC BEH C(18) column was used with 0.1% formic acid solution as the mobile phase under the condition of gradient elution. Preservatives were extracted with methanol by ultrasonicator, and then they were analysed by UPLC-PDA detector. All these preservatives were baseline separated in 8.5 min. The pre-treatment method of samples and the chromatographic condition of analysis were critically examined in this study. The recoveries ranged from 90.5 to 97.8%, with RSD values below 3.2%, and all correlation coefficients (r) were no less than 0.9997. Thus, this method could be used for analysing the preservatives in cosmetic products.

A rapid method for chemical fingerprint analysis of Hoodia species, related genera, and dietary supplements using UPLC–UV–MS

Recently, ultra-performance liquid chromatography (UPLC) has proven to be one of the most promising developments in the area of high-speed chromatographic separations with increased sensitivity and resolution. In this work, a reverse phase chromatographic method was developed using UPLC for the chemical fingerprint analysis of 12 hoodigosides, related genera and dietary supplements. The method is also used for the quantification of P57 in Hoodia species and dietary supplements that claim to contain Hoodia. The analysis was performed on a Waters Acquity UPLC system with an Acquity UPLC BEH C18 column (100 mm × 2.1 mm I.D., 1.7 μm) and a gradient elution of water and acetonitrile, both containing 0.05% formic acid with a run time of 15 min. The calibration curve of P57 showed good linearity ( r 2 > 0.999) within the established range (1–100 μg/mL). The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.3 and 0.9 μg/mL, respectively. The RSD for intra- and inter-day were less than 3.0%, and the recovery efficiency as 97–103%. LC–mass spectrometry coupled with electrospray ionization (ESI) interface method is described for the identification of P57. The developed method was successfully applied to the identification of 12 oxypregnane glycosides in four different species of Hoodia, 23 related genera and 35 dietary supplements that claim to contain H. gordonii. The UPLC profiles of various plant samples were compared for the presence of oxypregnane glycosides. Different sample matrices were successfully analyzed, providing the wide range of applicability of this method, including gels, capsules, tablets, sprays, tea bags, snack bars, powders and juices.

A rapid method for simultaneous determination of 15 flavonoids in Epimedium using pressurized liquid extraction and ultra-performance liquid chromatography

Herba Epimedii (family Berberidaceae), Yinyanghuo in Chinese, is one of commonly used Chinese medicines. Flavonoids are considered as its active components. In this study, a rapid ultra-performance liquid chromatography (UPLC) method was developed for simultaneous determination of 15 flavonoids, including hexandraside E, kaempferol-3- O-rhamnoside, hexandraside F, epimedin A, epimedin B, epimedin C, icariin, epimedoside C, baohuoside II, caohuoside C, baohuoside VII, sagittatoside A, sagittatoside B, 2″- O-rhamnosyl icariside II and baohuoside I in different species of Epimedium. The analysis was performed on Waters Acquity UPLC system with an Acquity UPLC BEH C18 column (50 mm × 2.1 mm I.D., 1.7 μm) and gradient elution of 50 mM acetic acid aqueous solution and acetonitrile within 12 min. All calibration curves showed good linearity ( R 2 > 0.9997) within test ranges. The LOD and LOQ were lower than 0.13 and 0.52 ng on column, respectively. The R.S.D.s for intra- and inter-day of 15 analytes were less than 5.0% at three levels, and the recoveries were 95.0–103.7%. The validated method was successfully applied to quantitatively analyze 15 flavonoids in different species of Epimedium. The results showed there were great variations among the contents of investigated flavonoids. Hierarchical clustering analysis based on characteristics of 15 investigated compounds peaks in UPLC profiles showed that 37 samples were divided into 3 main clusters, which were in accordance with their flavonoids contents. The simulative mean chromatogram of the high content cluster was generated to compare the samples from different species and/or locations of Epimedium. Four flavonoids including epimedin A, B, C and icariin were selected as markers for quality control of the species of Epimedium used as Yinyanghuo.

Fast simultaneous determination of 14 nucleosides and nucleobases in cultured Cordyceps using ultra-performance liquid chromatography

Determination of nucleosides and their metabolic compounds is important for physiological and pharmacological studies. Herein, a rapid ultra-performance liquid chromatography (UPLC) method was developed for the simultaneous determination of 14 nucleosides and nucleobases, namely adenine, adenosine, cytosine, cytidine, uracil, uridine, guanine, guanosine, hypoxanthin, inosine, thymine, thymidine, 2′-deoxyuridine and cordycepin. The separation was performed on Waters Acquity UPLC system with Acquity UPLC BEH C 18 column and gradient elution of 0.5 mM acetic acid and acetonitrile in 5 min. The correlation coefficients of 14 analytes were high ( R 2 > 0.9995) within the test ranges. The LOD and LOQ were lower to 11.9 and 47.0 ng/ml with 1 μl of injection volume, respectively. The overall R.S.D. for intra- and inter-day of 14 analytes were less than 1.8%. The developed method was applied for the analysis of nucleosides and nucleobases in cultured Cordyceps, which also could be used for the fast determination of the analytes in pharmaceutical products and biological fluids.