An effective high-performance liquid chromatography method for fingerprint and multi-component quantification of Oxalis corniculata

Objectives In this study, we aimed to compare modified Krauss polyacrylamide gradient gel electrophoresis (PAGGE) and high-performance liquid chromatography (HPLC) methods in classification, quantification, and separation of lipoproteins and determining low-density lipoprotein (LDL) size. Methods Blood specimens were obtained from eighty-seven volunteers. We measured LDL size using the PAGGE method and HPLC method with total cholesterol (TC) and triglyceride (TG) peaks. In the PAGGE method, Coomassie Brilliant Blue (CBB) staining was used instead of Sudan black staining, unlike the original method. The relationship between PAGGE and HPLC methods was evaluated by Pearson correlation test and Passing-Bablok regression analysis. Agreement between them was evaluated by Kappa analysis and Bland-Altman plots. Results Statistically significant correlation was found between the LDL size with PAGGE and HPLC methods under the cholesterol curve (HPLC-TC) (r=0.924, p<0.001). Similarly, there was a statistically significant correlation between PAGGE and HPLC methods under the TG curve (HPLC-TG) (r=0.910, p<0.001). In the PAGGE method, within-day precision was found as 2% and between-day precision as 3%. It was determined agreement between HPLC-TC vs. HPLC-TG methods and HPLC-TG vs. PAGGE methods was higher than HPLC-TC vs. PAGGE (Kappa values; 0.68, 0.71, and 0.44, respectively). Conclusions The PAGGE method can be a reliable method for measuring LDL size. HPLC method under cholesterol and triglyceride peaks may be used in clinical practice interchangeably, but clinical decision limits should be different. In addition, our study demonstrated that measurement methods for LDL size could be simplified with several modifications.

Objective To make comparative analysis of the glycated hemoglobin A1c(HbA1c) values obtained by three HbA1c test methods in patients with variant hemoglobins. Methods Total of 50 blood samples of patients with different types of variant hemoglobin were collected from January 2012 to December 2012; 25 of them (14 males and 11 females) carried hemoglobin D, Q, G, J and E with a mean age of (24±3) years; the other 25 cases (11 males and 14 females) were blood samples with hemoglobin F from newborn infants. Meanwhile, 50 blood samples(25 males and 25 females) from people without variant hemoglobins were also collected as control(mean age (25±5) years). Three methods were used to test HbA1c, which were affinity high performance liquid chromatography (HPLC) method (Ultra 2 of American Primus), ion exchange HPLC method (VariantⅡ of American Bio-Rad and G8 of Japanese Tosoh) and immunization method (DCA Vantage of German Siemens). The statistic analysis were done with variance analysis and correlation analysis. Results The HbA1c of the group with normal HbA1c structure was 5.7%±1.1%, 5.7%±1.2%, 5.7%±1.2% and 5.7%±1.1% respectively when it was tested by affinity HPLC method (Ultra2), ion exchange HPLC method (G8), ion exchange HPLC (Variant Ⅱ) and immunization method (DCA Vantage), and there was no significant difference among the groups (F=0.023, P>0.05). In the 25 samples with hemoglobin F, HbA1c could not be detected by ion exchange HPLC or immunoassay method. The fasting blood glucose level correlated with HbA1c level tested by Ultra 2 method (r=0.647, P<0.05), but it didn't correlate with HbA1c level when tested with VariantⅡ and G8 as well as DCA Vantage method. The HbA1c result of affinity HPLC method was free from the disturbance of Hb D, Q, G, J and E, and had a obvious correlation with blood glucose (r=0.823, P<0.05). The HbA1c result of ion exchange HPLC method was disturbed by hemoglobin D, Q, G, J and E in varying degrees. The HbA1c measured by immunization method was associated with blood glucose (r=0.611, P<0.05). Conclusion The HbA1c value obtained by affinity HPLC method can accurately reflect the mean blood glucose level. Variant hemoglobins disturb the HbA1c result tested by ion exchange HPLC method, and the immunization test result is only disturbed by hemoglobin F. Key words: Glycated hemoglobin A1c; Variant hemoglobins; High performance liquid chromatography

Decaffeinated coffee accounts for 10 percent of coffee sales in the world; it is preferred by consumers that do not wish or are sensitive to caffeine effects. This article presents an analytical comparison of capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) methods for residual caffeine quantification in decaffeinated coffee in terms of validation parameters, costs, analysis time, composition and treatment of the residues generated, and caffeine quantification in 20 commercial samples. Both methods showed suitable validation parameters. Caffeine content did not differ statistically in the two different methods of analysis. The main advantage of the high performance liquid chromatography (HPLC) method was the 42-fold lower detection limit. Nevertheless, the capillary electrophoresis (CE) detection limit was 115-fold lower than the allowable limit by the Brazilian law. The capillary electrophoresis (CE) analyses were 30% faster, the reagent costs were 76.5-fold, and the volume of the residues generated was 33-fold lower. Therefore, the capillary electrophoresis (CE) method proved to be a valuable analytical tool for this type of analysis.

Inulin and oligofructose exhibit valuable nutritional and functional attributes, and so the utilization of inulin and its derivatives in the food industry has been continuously increasing. Compared with high-performance liquid chromatography (HPLC) which presents improved sensitivity and precision, colorimetric method is more widely used and accepted due to the advantages of its simplicity and economy. In order to determine inulin for the purpose of labeling, we develop an easy and effective HPLC method after extraction of inulin. HPLC conditions include a Shim-pack SCR 101C column (Shimadzu), deionized water at 80 °C as the mobile phase, and a refractive index detector (RID). Moreover, we compared the HPLC method and colorimetric method on tested foods including claimed commercial inulin products such as inulin, inulin oligomers, and commercial food products. The obtained results demonstrated that the HPLC method could avoid the overestimation and inaccuracy caused by colorimetric method in the case where the samples contain glucan or sucrose, and be regarded as an approach which is easier, faster, and more accurate than colorimetric method for the routine quantification of inulin and inulin oligomers in commercial products.

PurposeThe Pharmacy Service of the Infanta Leonor University Hospital acquires, compounds, distributes and dispenses more than 3000 L of methadone oral solution to Drug Addiction Patients Centers per year. Our purpose is to develop and validate an improved high performance liquid chromatography (HPLC) method to quantify methadone hydrochloride in a new oral solution with methylhydroxybenzoate (methylparaben) and propylhydroxybenzoate (propylparaben) to be implemented in physicochemical stability studies that allow to provide more information and even to increase the beyond-use date.MethodsA HPLC-Agilent® 1100 equipment, comprising a quaternary pump and an ultraviolet diode-array-detector (DAD) was used. An analytical method development and validation was completed. The curve was constructed from methadone working concentrations of 75–125% (7.5, 9.0, 10.0, 11.0 and 12.5 mg/mL) to assess the linear relationship between the concentration of the analyte and the obtained areas. Precision and accuracy were calculated. Detection and quantification limit (LD, LQ) were estimated using the EURACHEM method. Forced-degradation studies were also performed.ResultsChromatographic conditions were: flow rate 1.6 mL/min; mobile phase 55% acetonitrile and 45% sodium phosphate 25 mM (pH = 10); injection volume was 5 µL. The column was a Waters-XTerra™ RP18, maintained at 40 °C. DAD was λ = 254 nm. Retention times for methadone, methylparaben and propylparaben were 4.34, 0.70 and 0.88 min respectively.The method was linear (y = 284.3x − 97.8, r = 0.996). Instrumental precision was 0.33% for standards (n = 10); intra-assay precision 0.53% (n = 6) and inter-assay precision 1.95% (n = 12). The relative standard deviation percentage for accuracy was 1.28%. The recovery percentage was 101.5 ± 1.5%. LQ and LD were 2.18 µg/mL and 2.0 µg/mL respectively. The most destabilizing conditions were oxidizing and alkaline. The chromatograms confirmed no interference with the methadone signal.ConclusionsThe HPLC method has proved to be valid and reproducible for methadone quantification in a new oral solution with methylparaben and propylparaben. This assay is a rapid, simple and reliable technique that can be used in daily analysis and physicochemical stability studies.

The aim of this study was to optimize a high performance liquid chromatography (HPLC) method for the simultaneous quantification of the four main compounds geniposide, berberine, baicalin and crocin present in the Traditional Chinese formula Huanglianjiedutang (HLJDT). This formula contains Coptidis Rhizoma, Radix Scutellariae, Cortex Phellodendri and Fructus Gardeniae in a ratio of 1.5: 1: 1: 1.5. The decoction of the formula was prepared in the traditional Chinese way. The most suitable boiling time for the decoction turned out to be 60 min. Validation of the method was performed to demonstrate its reproducibility, precision, stability, repeatability, and recovery rate. Excellent behaviours over the investigated concentrations were observed with the values of RSD ≤ 5%. For the estimation of the recovery rate, half of the herbs were boiled together with the pure compounds. As crocin turned out to be an unstable compound its recovery rate could not be determined. The optimized HPLC method provides an accurate and reproducible analysis for the simultaneous determination of geniposide, berberine, baicalin, and crocin in the formulation HLJDT which is a very important tool for the quality control of this Traditional Chinese Medicine (TCM) formula. Key words: High performance liquid chromatography (HPLC), quantification, validation, huanglianjiedutang (HLJDT), geniposide, berberine, crocin, baicalin.

BackgroundThe introduction of a GMP-certified 68Ga-generator spurred the application of 68Ga-radiopharmaceuticals. Several radiosynthesis of 68Ga-radiopharmaceuticals are more efficient and robust when performed with 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (HEPES) buffer, which is considered as an impurity in the quality control (QC) procedure. Thus, prior to clinical use, QC must be conducted to ensure that HEPES does not exceed the maximum dose of 200 μg/V Injected as described in European Pharmacopoeia (Ph Eur) for edotreotide. However, when applying the thin-layer chromatography (TLC) method described in the Ph Eur to quantify the HEPES amount present in the 68Ga-octreotide or in the remaining 68Ga-radiopharmaceuticals that were tested, no amount was detectable after 4 min of iodine incubation. Here we tested our modified TLC method and validate a new high-performance liquid chromatography (HPLC) method to quantify HEPES in 68Ga-radiopharmaceuticals and compare it to the TLC-method described in Ph Eur. In addition, samples collected from various institutes were tested to evaluate whether the synthesis of different 68Ga-radiopharmaceuticals or the use of different synthesis methods could affect the amounts of HEPES.ResultsHEPES could not be detected by the TLC method described in the Ph Eur within 4 min incubation in an iodine-saturated chamber. As for our modified TLC method, only after 2 h, spots were only visible > 1 mg/mL. The HPLC method had a limit-of-quantification (LOQ) of 3 μg/mL and a limit-of-detection (LOD) of 1 μg/mL. From the three 68Ga-radiopharmaceuticals tested, only in the [68Ga]Ga-NODAGA-Exendin samples exceeding amounts of HEPES were found and its concentration in the [68Ga]Ga-NODAGA-Exendin was significantly higher, when compared to [68Ga]Ga-DOTATOC and [68Ga]Ga-PSMA-11.ConclusionThe TLC method described in Ph Eur and our modified TLC method may not be sufficiently sensitive and thus unsuitable to use for QC release. The new HPLC method was sensitive, quantitative, reproducible and suitable for QC release. With this method, we were able to determine that some 68Ga-radiopharmaceuticals may exceed the HEPES limit of 200 μg/ V Injected. This new analytical system would allow correcting for the maximum injected dose in order not to exceed this amount.

A simple and rapid HPLC/UV method for the simultaneous quantification of theophylline and etofylline in human plasma

Background: Achieving a balanced mixture with the right preservative concentration is most crucial part to maintain the stability of a product and its shelf life. Objective: The objective is to employ a sensitive method for accurate determination of all preservatives namely, bronopol, sodium benzoate, methylparaben, and propylparaben in antitussive kid drops (Zeal Kid Drops). Materials and Methods: The method was successfully developed and validated with the reference to the International Council for Harmonization (ICH) guidelines. All the analytes were separated within 30 min on column C18 (5 μm, 4.6 mm × 250 mm) using mobile phase; phosphate buffer: acetonitrile (65:35) at 40°C, in isocratic mode, and flow rate was 1 mL/min. Method validation was assessed as per ICH Q2(R2) guideline. Results: Bronopol, sodium benzoate, methylparaben, and propylparaben were quantified, and percentage of preservatives was 0.015, 0.483, 0.201 and 0.037, respectively, using developed method. Specificity, linearity, accuracy, precision, robustness, limit of detection, and limit of quantification were established and found within the acceptable range. Conclusion: The developed Reverse Phase - High Performance Liquid Chromatography (RP-HPLC) method is simple, precise, and accurate; making it suitable for utilization as a quality control tool for liquid oral formulations.

The topical veterinary drug product containing fipronil and permethrin provides an effective repellent protection and high insecticidal efficacy for dogs. The objective of this study was to develop a stability-indicating high-performance liquid chromatography (HPLC) method for simultaneous detection and quantification of fipronil, permethrin, their key degradation products, and butylated hydroxytoluene (BHT) in a topical drug product. The two active ingredients, their degradation products, and the antioxidant (BHT) were separated by a gradient elution on a Phenomenex Kinetex C18 column (150 × 3 mm, 2.6 µm particle size) maintained at 37°C with H2O acetonitrile isopropyl alcohol 85% H3PO4 (65.5 + 32.5 + 4/0.0053, v/v/v/v) as mobile phase A and acetonitrile (100%) as mobile phase B. The flow rate was 0.9 mL/min, and analytes were detected and quantified at 235 nm. The specificity of the method was demonstrated by adequate separation of fipronil, permethrin, their degradation products, and BHT in the forced degraded finished product. The linearity of the method was demonstrated in the range of 0.2% to 150% of target analytical concentration of both active ingredients and 50% to 150% for BHT. Excellent recoveries of fipronil, permethrin, and BHT in placebo spiked active ingredient solutions in the linearity range showed sufficient accuracy of the method. The LOQ and LOD of the method were determined to be 0.2% and 0.07% of the analytical concentration. A robustness study did not identify any critical parameter that adversely affected the separation and quantification. Here, we report the development and validation of a robust, stability-indicating HPLC method for identification and assay of fipronil, permethrin, and BHT, including estimation of fipronil's and permethrin's degradation products in a topical drug product for dogs. The new HPLC method permits the acquisition of data for all analytes of interest for a topical finished drug product containing fipronil, permethrin, and BHT.

Background: The 99mTc-tetrofosmin is a radiopharmaceutical used in oncology for scintigraphic quantification of the myocardial perfusion. The preparation of this drug is based on a complexation reaction of technetium 99 metastable (99mTc) with tetrofosmin. The reference method for quality control is thin layer chromatography, using TLC SA bands. This method is simple but only separates two types of impurities: free technetium and hydrolyzed technetium associated to hydrophilic impurities like gluconate-99mTc and takes from 30 to 35 minutes. This gluconate impurity gives a poor image quality and difficult interpretation issues. HPLC is a sensitive and specific method, it thus, has an interest in controlling RCP and identifying all impurities. Methods: The reference method is a method by planar chromatography TLC SA tape, size 1 cm x 20 cm. Two marks were scored: 3 cm from the edge to indicate the deposit (10 µL of the preparation) and 15 cm by the end of migration. Mobile phase was acetone: dichloromethane (65:35, v/v). The radioactive bands were quantified by counting the radioactivity using a radiochromatograph miniGITA® (Raytest) equipped with a scintillation probe. The chromatographic system consisted in a Symmetry Shield® column RP18 5μm 100Å (Waters) with a gamma detector Gammaram® (Lablogic). Empower® software (Waters) was used for peak integration. The mobile phase, at a rate flow of 1.0 mL / min, consisted of a mixture of acetonitrile (Waters) and titrisol® buffer (Waters) (40:60, v/v). The sample volumes injected were no more than 10 to 30 µL in order not to exceed 50,000 counts per second due to the risk of radioactive detector saturation. Results: The RCP was measured simultaneously by HPLC and reference method in 30 preparations. For HPLC, mean RCP = 97.21%, α= 2.178% [91.6%-99.63%]. For TLC SA, mean RCP = 97.99%, α= 1.135% [94.31%-99.86%]. The results obtained by both methods were compared using the Wilcoxon t test. The RCP obtained either by TLC SA or HPLC methods are not significantly different (p-value = 0,497, higher than in significance ≤ = 0.05) Conclusions: A new HPLC method was developed for the control of the RCP 99mTc-Tetrofosmin. This method is reliable, rapid, sensitive and easy to use when the equipment is available. It allows us to improve the detection of cardiotoxic side effects due to chemotherapy more quickly than TLC SA method and to prevent toxicity by dose adjustment of anticancer drugs. Although the HPLC method does not differ from TLC (reference method), HPLC provides additional information about the quality of the preparation (percentage of gluconate-99mTc). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2451. doi:1538-7445.AM2012-2451

Comparison of column-coupled electrophoresis with liquid chromatography methods in food analysis of quinineComparison of column-coupled electrophoresis with liquid chromatography methods in food analysis of quinine (QUI) is presented in this work. The capillary isotachophoresis (CITP) on-line coupled with capillary zone electrophoresis (CZE) and hyphenated with fibre-based spectrophotometric diode array detection (DAD) was compared with, (i) high performance liquid chromatography (HPLC) method with DAD detection, and (ii) HPLC method with fluorescence detection (FD). These methods were compared through their performance parameters and determined concentrations of QUI in beverages. The concentrations of QUI in two selected bitter drinks determined by the CITP-CZE-DAD method were in a good accordance with the HPLC-DAD and HPLC-FD methods. In addition, the electrophoretic method, as well as the chromatographic methods, was able to separate potential QUI related impurities from the QUI peak. The CITP-CZE-DAD method provided excellent performance parameters that were comparable (precision, accuracy, LOD, robustness) or even better (separation efficiency) than those ones provided by the chromatographic methods. Moreover, the effectivity of the electrophoresis method was higher when considering cost of analysis (equipment, consumption of separation systems), environmental aspects (organicvs. aqueous solvents), on-line sample pretreatment (CITP preconcentration and sample clean-up suitable also for the more complex matrices). Considering these findings, CITP-CZE-DAD was approved as a routine automatized method for the highly reliable quality food control.

Purpose : To develop and validate a simple ultrafast monolithic high performance liquid chromatography (HPLC) method for the simultaneous quantification of two anti-cancer agents, imatinib and sorafenib, in pure form and tablet preparations. Method : Chromatographic separation was accomplished using Chromolith flash RP-18 HPLC-column (25 - 4.6 mm; macropores, 2 μm; mesopores, 13 – 15 nm). The optimum mobile phase composition of ammonium acetate buffer (10 mM, pH 8.5) and methanol at ratio of 35:65 v/v was used. Effluent flow rate was adjusted to 1.0 mL/min and the analysis was performed at 250 nm wavelength. The developed method was evaluated for specificity, linearity, precision and accuracy. Results : The method offered a linear relationship over the concentration range of 1 - 16 μg/ml (correction coefficient, R 2 = 0.9999) for both analytes. Limit of detection (LOD) was 0.1891 and 0.1888 μg/ml while limit of quantification (LOQ) was 0.6303 and 0.6294 μg/ml for imatinib and sorafenib, respectively. Mean recovery was within 100 ± 2 %. The utility of the new method was demonstrated by its successful use for the analysis of commercially available tablet formulations of both drugs. Conclusion : The developed method is fast and economical, and is being recommended for routine analysis of imatinib and sorafenib in bulk drug and tablet dosage forms in quality control laboratories. Keywords : RP-HPLC, Chromolith, Imatinib, Sorafenib, Validation, Quality control

A combination method of multi-wavelength fingerprinting and multi-component quantification by high performance liquid chromatography (HPLC) coupled with diode array detector (DAD) was developed and validated to monitor and evaluate the quality consistency of herbal medicines (HM) in the classical preparation Compound Bismuth Aluminate tablets (CBAT). The validation results demonstrated that our method met the requirements of fingerprint analysis and quantification analysis with suitable linearity, precision, accuracy, limits of detection (LOD) and limits of quantification (LOQ). In the fingerprint assessments, rather than using conventional qualitative “Similarity” as a criterion, the simple quantified ratio fingerprint method (SQRFM) was recommended, which has an important quantified fingerprint advantage over the “Similarity” approach. SQRFM qualitatively and quantitatively offers the scientific criteria for traditional Chinese medicines (TCM)/HM quality pyramid and warning gate in terms of three parameters. In order to combine the comprehensive characterization of multi-wavelength fingerprints, an integrated fingerprint assessment strategy based on information entropy was set up involving a super-information characteristic digitized parameter of fingerprints, which reveals the total entropy value and absolute information amount about the fingerprints and, thus, offers an excellent method for fingerprint integration. The correlation results between quantified fingerprints and quantitative determination of 5 marker compounds, including glycyrrhizic acid (GLY), liquiritin (LQ), isoliquiritigenin (ILG), isoliquiritin (ILQ) and isoliquiritin apioside (ILA), indicated that multi-component quantification could be replaced by quantified fingerprints. The Fenton reaction was employed to determine the antioxidant activities of CBAT samples in vitro, and they were correlated with HPLC fingerprint components using the partial least squares regression (PLSR) method. In summary, the method of multi-wavelength fingerprints combined with antioxidant activities has been proved to be a feasible and scientific procedure for monitoring and evaluating the quality consistency of CBAT.

Larotaxel is a new chemical structure drug, which has not been marketed worldwide. Accordingly, the standard identification and quantification methods for larotaxel remain unclear. The spectrometric analyses were performed for verifying weight molecular formula, molecular weight and chemical structure of larotaxel. Besides, a quantification method was developed for measuring larotaxel in the liposomes. The molecular formula, molecular weight and chemical structure of larotaxel were studied by using mass spectrometry (MS), infra-red (IR), nuclear magnetic resonance (NMR) and ultraviolet-visible (UV-vis) spectrometric techniques. The absorption wavelength of larotaxel was investigated by UV-vis spectrophotometry full-wavelength scanning. Besides, a quantification method was developed by high performance liquid chromatography (HPLC), and then validated by measuring the encapsulation efficacy of larotaxel liposomes. The four spectral characteristics of larotaxel were revealed and the corresponding standard spectra were defined. It was confirmed that larotaxel had the structure of tricyclic diterpenoids, with the molecular formula of C45H53NO14, the molecular weight of 831.900 1, and the maximum absorption wavelength of 230 nm. The quantitative method of larotaxel was established by using HPLC with a reversed phase C18 column (5 μm, 250 mm×4.6 mm), a mobile phase of acetonitrile-water (75:25, volume/volume), and a detection wavelength of 230 nm. The validation study exhibited that the established HPLC method was stable, and had a high recovery and precision in the quantitative measurement of larotaxel in liposomes. In addition, a new kind of larotaxel liposomes was also successfully prepared. The particle size of the liposomes was about 105 nm, with an even size distribution. And the encapsulation efficiency of larotaxel in the liposomes was above 80%. The present study offers reference standard spectra of larotaxel, including MS, IR, NMR, and UV-vis, and confirms the molecular formula, molecular weight and chemical structure of larotaxel. Besides, the study develops a rapid HPLC method for quality control of larotaxel liposomes.