ultra-HPLC Method Research Articles

Determination of Phthalic Acid, 2-(3',5'-Dibromo-2',4'-Dihydroxybenzoyl) Benzoic Acid, and Tribromoresorcinol in the Color Additives D&C Red No. 21, D&C Red No. 22 (Eosin Y), and Their Lakes Using UHPLC.

Background: Certain impurities in the color additives drug and cosmetic (D&C) Red No. 21 (R21), D&C Red No. 22 (R22), and their lakes are limited to levels specified in the Code of Federal Regulations (CFR) and are quantified by the U.S. Food and Drug Administration in batches of these color additives submitted for certification. Currently, a lengthy and tedious method based on gravity flow elution column chromatography is used to quantify the following CFR-specified impurities: the intermediate, phthalic acid (PhthAc); the manufacturing by-products, 2-(3',5'-dibromo-2',4'-dihydroxybenzoyl)benzoic acid (Br2BBA); and brominated resorcinol. "Brominated resorcinol" implies the sum of all possible brominated resorcinols, but the current work focused on 2,4,6-tribromoresorcinol (Br3R) as the most probable side-reaction product. Objective: An improved method was needed to quantify PhthAc, Br2BBA, and Br3R in R21, R22, and their lakes. Methods: A rapid ultra-HPLC (UHPLC) method was developed to replace the gravity flow method for quantitative determination of PhthAc, Br2BBA, and Br3R. Results: PhthAc, Br2BBA, and Br3R were quantified by using five-point calibration curves with data point ranges of 0.11-1.55, 0.06-0.77, and 0.04-0.61% by weight, respectively. LODs for the analytes ranged from 0.01 to 0.03%. Recoveries of the analytes ranged from 90.6 to 99.9%. Conclusions: The UHPLC method is accurate and significantly more rapid than the gravity -flow method, requiring approximately 7 min as compared with 6 h to detect PhthAc, Br2BBA, and Br3R in one sample. Highlights: A rapid UHPLC method was developed to determine CFR-specified impurities in the color additives D&C Red Nos. 21 and 22 and their lakes.

Determination of Aniline, 4-Aminoazobenzene, and 2-Naphthol in the Color Additive D&C Red No. 17 Using Ultra-High-Performance Liquid Chromatography.

Specifications in the U.S. Code of Federal Regulations for the color additive D&C Red No. 17 (R17, Colour Index No. 26100) limit the levels of the dye's intermediates, aniline (AN), 2-naphthol (β-naphthol, BN), and 4-aminoazobenzene (4AAB), to 0.2, 0.2, and 0.1%, respectively. The present work reports the development and application of an ultra-HPLC method for the quantitative determination of these impurities in R17. A 1.7 μm particle size C-18 column was used with 0.2 M ammonium acetate and acetonitrile as the eluents. AN, BN, and 4AAB were quantified by using six-point calibration curves with data points (w/w) ranging from 0.01 to 0.25% for AN, 0.01 to 0.24% for BN, and 0.01 to 0.19% for 4AAB. The correlation coefficients ranged from 0.9992 to 0.9999. Limits of detection for the analytes ranged from 0.002 to 0.01%. Recoveries of the analytes ranged from 99.5 to 102%. Relative standard deviations ranged from 0.482 to 1.262%. The new method was applied to analyze portions from 22 batches of R17 submitted to the U.S. Food and Drug Administration for certification. It was found to be simpler to implement, faster, and more sensitive than the older gravity-elution column chromatography method, which it has replaced.

Ultra HPLC Method for Fixed Dose Combination of Azilsartan Medoxomil and Chlorthalidone: Identification and in silico Toxicity Prediction of Degradation Products

The fixed dose combination of azilsartan medoxomil potassium and chlorthalidone has been introduced for the effective treatment of hypertension. In the present work a rapid, simple and accurate stability indicating ultra HPLC assay method has been developed. The separation of azilsartan medoxomil, chlorthalidone and their degradation products were accomplished on an Acquity UPLC BEH C18 (100 mm × 2.1 mm, 1.7 μm) column using mobile phase combination of 0.02% trifluoroacetic acid in water and acetonitrile in gradient mode. The forced degradation products were identified using liquid chromatography‒electrospray ionisation-quadrupole time of flight-tandem mass spectrometry (LC‒ESIQTOF–MS/MS) and accurate mass experiments. The in silico toxicities of the degradation products for both the drugs were evaluated. The proposed method was validated as per the ICH Q2 (R1) guideline for selectivity, linearity, precision, accuracy and robustness.

Determination of Lutein and β-Carotene in Infant Formula and Adult Nutritionals by Ultra-High-Performance Liquid Chromatography: Single-Laboratory Validation, First Action 2016.13

An ultra-HPLC method for the determination of lutein and β-carotene in infant formula and adult nutritionals was validated using both unfortified and fortified samples provided by the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN). All experiments showed separation of all-trans-lutein and β-carotene from their major cis isomers, apocarotenal, α-carotene, lycopene, and zeaxanthin. Samples spiked with all-trans-lutein and β-carotene showed no isomerization during sample preparation. Linearity of the calibration solutions correlated to approximately 0.8-45 μg/100 g (reconstituted basis) for samples prepared for the lowest sample concentrations. With dilutions specified in the method, the range can be extended to approximately 2250 μg/100 g. The LOD for both lutein and β-carotene was 0.08 μg/100 g, and the LOQ for both was 0.27 μg/100 g. For all measurements in the range of 1-100 μg/100 g, repeatability RSD was ≤5.8% for lutein and ≤5.1% for β-carotene. For measurements >100 μg/100 g, repeatability RSD was ≤1.1% for lutein and ≤1.7% for β-carotene. Accuracy was determined by recovery from spiked samples and ranged from 92.3 to 105.5% for lutein and from 100.1 to 107.5% for β-carotene. The data provided show that the method meets the criteria specified in the Standard Method Performance Requirements for carotenoids (SMPR 2014.014).

Ultra-HPLC method for quality and adulterant assessment of steviol glycosides sweeteners – Stevia rebaudiana and stevia products

Stevia products are advertised as a zero-calorie sweetener. Glucose should not be an intrinsic component of this product, but it has been identified from some of stevia products in a preliminary study. An UHPLC-UV method was developed for the quantitative determination of glucose from stevia products. After stevia products reacted with 1-phenyl-3-methyl-5-pyrazolone (PMP), PMP derivatives were analysed and glucose was found in seven out of 35 products in the range 0.3–91.5% (w/w). Two products, SPR-12 and SPR-27, showed remarkable amounts of glucose at 61.6% and 91.5%, respectively. In addition, an UHPLC-UV-evaporative light-scattering detector (ELSD) method was developed for the quantitative determination of rebaudioside A, stevioside, rebaudioside D, dulcoside A and steviolbioside from Stevia rebaudiana and related products. In a 12 min run, five steviol glycosides were baseline-separated. ELSD and ultraviolet (UV) detections showed comparable results. The LC methods were validated for linearity, repeatability, accuracy, limits of detection (LOD) and limits of quantification (LOQ). For steviol glycosides, the LODs and LOQs were found to be less than 10 and 30 μg ml–1, respectively. The RSD for intra- and inter-day analyses was less than 2.5%, and the recovery was 90–94%. For PMP derivative of glucose, the LOD and LOQ were 0.01 and 0.05 μg ml–1, respectively. Repeatability (RSD) was less than 2.6%; recovery was 98.6–101.7%. The methods are useful for the identification, quality assurance, and adulterant assessment of S. rebaudiana and steviol glycosides sweeteners (stevia products).

Determination of total ginsenosides in ginseng extracts using charged aerosol detection with post-column compensation of the gradient

AimVariation in structure-related components in plant products prompted the trend to establish methods, using multiple or total analog analysis, for their effective quality control. However, the general use of routine quality control is restricted by the limited availability of reference substances. Using an easily available single marker as a reference standard to determine multiple or total analogs should be a practical option. MethodIn this study, the Ultra-HPLC method was used for the baseline separation of the main components in ginseng extracts. Using a plant chemical component database, ginsenosides in ginseng extracts were identified by Ultra-HPLC-MS analysis. The charged aerosol detection (CAD) system with post-column compensation of the gradient generates a similar response for identical amounts of different analytes, and thus, the content of each ginsenoside in ginseng extracts was determined by comparing the analyte peak area with the reference standard (determination of total analogs by single marker, DTSM). The total ginsenoside content was determined by the summation of reference standard and other ginsenoside components. ResultsThe results showed that DTSM approaches were available for the determination of total ginsenosides in a high purity ginseng extract because of the removal of impurities. In contrast, DTSM approaches might be suitable for determination of multiple ginsenosides without interference from impurities in the crude ginseng extract. ConclusionFuture practical studies similar to the present study should be conducted to verify that DTSM approaches based on CAD with post-column inverse gradient for uniform response are ideal for the quality control of plant products.

Validation and clinical application of an UHPLC method for simultaneous analysis of total homocysteine and cysteine in human plasma

Several studies indicate that high levels of homocysteine (Hcy) and L-cysteine (L-Cys) are independent risk factors for cardiovascular disease. The validation and clinical application of an ultra HPLC method for analysis of Hcy and L-Cys is described. The reported method is simple, sensitive, rapid, precise, and less aggressive than other previously reported methods. The effect of the derivatization reaction time, pH, and organic solvent contents in the mobile phase are described and discussed. Optimized conditions resulted in excellent peak shapes. Results of method validation showed a good linearity (r(2) ≥ 0.993) over the investigated concentration ranges and were observed for both compounds. The LOD and LOQ were 0.05 μM and 0.15 μM for Hcy and 0.24 μM and 0.80 μM for L-Cys, respectively. Validation results proved that the method precision was good and the accuracy was satisfactory. This validated method was successfully applied in an epidemiological study to measure and compare the prevalence of Hcy and L-Cys high levels in plasma of Portuguese type 2 diabetic patients with and without angiopathy. The study results showed that prevalence of hyperhomocysteinemia and hypercysteinemia were at least two times higher in diabetic patients with angiopathy compared to diabetics without angiopathy.

ULTRA HPLC METHOD FOR THE SIMULTANEOUS ANALYSIS OF DRUGS AND FLAVONOIDS IN HUMAN URINE

A validated reverse-phase Ultra HPLC method for the simultaneous determination of drugs sotalol, metoprolol, propranolol, carvedilol, salicylic acid, dexamethasone, prednisolone, and ketoprofen and flavonoids: (±)-catechin, (−)-epicatechin, rutin, hesperidin, neohesperidin, quercitrin, (±)-naringenin, hesperetin in human urine has been developed. Urine samples were pretreated by solid-phase extraction using SDB and C18 cartridges. The extraction efficiencies of each analyte from urine ranged from 76.21% to 101.29%. Gradient separation is achieved by using a Chromolith® Fast Gradient Monolithic C18e (50 mm × 2 mm) column and Hypersil Gold (50 mm × 2.1 mm, 1.9 µm) column, using UV detection to monitor the analytes at 227, 240, 254, and 280 nm. The mobile phase consists of mixed 0.05% trifluoroacetic acid in water and acetonitrile in the gradient elution. All sixteen compounds were analyzed within 4 min. The LOD and LOQ of drugs and flavonoids are 0.01 µg/mL and 0.03 µg/mL; 0.02 µg/mL and 0.07 µg/mL, respectively. The developed procedure allows the determination of drugs in urine in concentrations from 0.2 to 40 µg/mL. The following concentrations of the examined drugs in human urine, belonging to people treated with β-blockes, were detected. What is more, an amount of flavonoids occur in urine in higher concentrations than calculated LOD and LOQ. The method has been proved to be precise, accurate, and well suited to the routine determination of all these flavonoid and drug concentrations in human urine samples.

Fast HPLC–ECD analysis of ascorbic acid, dehydroascorbic acid and uric acid

A robust and rapid high-pressure liquid chromatography–electrochemical detection (HPLC–ECD) method was developed and validated for the accurate determination of ascorbic acid (AA) and uric acid (UA), in human plasma. Dehydroascorbic acid (DHAA) was indirectly measured by subtracting native ascorbic acid from total ascorbic acid concentrations; the latter was obtained after chemical reduction. A stable electrochemical active internal standard (homogentisic acid) was added for the accurate quantification of the analytes. The analyses were performed on a reverse-phase column with traditional HPLC and ultra-HPLC (UHPLC). The UHPLC method showed increased sensitivity with detection limit of 0.05 ng for both AA and UA, 2 times lower compared to conventional HPLC. UHPLC also reduced run times fourfold with less waste generation. Both assays showed good accuracy and precision, the intra- and inter-day CVs of AA and UA analysis are less than 7%.

Validated Ultra HPLC Method for the Simultaneous Determination of Atorvastatin, Aspirin, and their Degradation Products in Capsules

An Ultra HPLC method was developed and validated for the simultaneous determination of atorvastatin, aspirin, and their major degradation products in capsules. Salicylic acid, acetylsalicylsalicylic acid, salicylsalicylic acid, and the lactone form of atorvastatin were separated along with six unknown degradation products within 4 min. The chromatographic separation was performed on acquity UPLCTM BEH C18 column (1.7 µm, 2.1 × 50 mm); using a gradient elution of acetonitrile and phosphate buffer (0.01 M, pH 2.0) at a flow rate of 0.6 mL/min. UV detection was performed at 247 nm. The method was validated for accuracy, repeatability, reproducibility, and robustness. Stability indicating capability was established by forced degradation experiments and separation of known degradation products. Linearity, LOD, and LOQ was established for atorvastatin, aspirin, and their known degradation products.