Ultra-high Performance Liquid Chromatography Method Research Articles

Microbial inactivation at high flow velocity using carbon dioxide as a novel approach for rapid non-thermal decontamination in food biotechnology and bioprocess engineering

The relevance of high-throughput metabolomic analysis in plant sciences has increased, and has become a valuable stratagem for advancing plant genomics and investigating biochemical aspects of plants. There are diverse instrumental tools for accomplishing these tedious tasks in a limited time span, including spectroscopic methods such as proton nuclear magnetic resonance (1H-NMR) spectroscopy, gas chromatography mass spectrometry (GC-MS), and liquid chromatography mass spectroscopy (LC-MS). Determination of metabolites in bulk amounts can be carried out by (1H-NMR) spectroscopy; while GC-MS is applicable for volatile metabolites, and LC-MS is highly applicable for analyzing an extensive range of semi-polar metabolites comprised of valuable secondary metabolites. Recent advances in separation techniques have led to the discovery of ultra-high performance liquid chromatography (UHPLC) and MS-based technology methods exhibiting an amplified resolution of metabolites, and accuracy of mass estimations up to the sub-ppm accuracy level. This chapter is intended to provide readers with basic information on these techniques. Their improvement in recent years will highlight their new relevance in investigating plant metabolomic changes in response to ambient environmental and climate changes. Integration of chromatographic techniques with sensitive detectors can determine even meager variations in metabolite flux, and provide a snapshot of plant metabolomes.

Extraction of valuable compounds from microalgal biomass. Operating conditions and yield

The relevance of high-throughput metabolomic analysis in plant sciences has increased, and has become a valuable stratagem for advancing plant genomics and investigating biochemical aspects of plants. There are diverse instrumental tools for accomplishing these tedious tasks in a limited time span, including spectroscopic methods such as proton nuclear magnetic resonance (1H-NMR) spectroscopy, gas chromatography mass spectrometry (GC-MS), and liquid chromatography mass spectroscopy (LC-MS). Determination of metabolites in bulk amounts can be carried out by (1H-NMR) spectroscopy; while GC-MS is applicable for volatile metabolites, and LC-MS is highly applicable for analyzing an extensive range of semi-polar metabolites comprised of valuable secondary metabolites. Recent advances in separation techniques have led to the discovery of ultra-high performance liquid chromatography (UHPLC) and MS-based technology methods exhibiting an amplified resolution of metabolites, and accuracy of mass estimations up to the sub-ppm accuracy level. This chapter is intended to provide readers with basic information on these techniques. Their improvement in recent years will highlight their new relevance in investigating plant metabolomic changes in response to ambient environmental and climate changes. Integration of chromatographic techniques with sensitive detectors can determine even meager variations in metabolite flux, and provide a snapshot of plant metabolomes.

Quantitative ultra-high-performance liquid chromatography-tandem mass spectrometry for determination of dexmedetomidine in pediatric plasma samples: Correlation with genetic polymorphisms.

Dexmedetomidine is an important sedative agent administered as premedication to achieve procedural sedation in children. To describe the correlation between the genetic state and the concentration of dexmedetomidine, it is necessary to develop a specific, time-saving and economical method for detection of dexmedetomidine in plasma samples. In this work, an ultra-high-performance liquid chromatography (UHPLC)-tandem mass spectrometry method has been established and validated for detection of dexmedetomidine in plasma from pediatric population. After a simple liquid-liquid extraction with an organic solution, the analytes were separated on an ACQUITY BEH C18 column (2.1 mm × 50 mm, 1.7 μm particle size) by gradient elution with the mobile phase of acetonitrile and 1‰ aqueous formic acid (flow rate 0.3 mL min-1 ). Mass spectrometry measurements were performed under the positive selected reaction monitoring and the mass transitions monitored were m/z 201.3 → 95.1, 204.2 → 98.0 for dexmedetomidine and deuterated medetomidine (internal standard), respectively. Validation of the method based on China Food and Drug Administration guidelines showed acceptable selectivity. The UHPLC method employed a stable isotope-labeled internal standard, showed good specificity and was successfully used to detect dexmedetomidine in plasma samples from 260 pediatric patients. A subsequent application of this method to a pharmacogenetic study was also described. Importantly, this is the first study to report the correlation between CYP2A6 rs835309 activity and concentration of dexmedetomidine.

Development of a fast UHPLC-MS/MS for the analysis of selected priority micropollutants in wastewater samples

ABSTRACT In recent years, there have been many reports on the presence of organic compounds in different compartments of the aquatic environment. The subject of our research was the development of a new method for the determination of selected organic micropollutants and their transformation products in wastewater samples. A rapid and sensitive ultra-high performance liquid chromatographic method coupled with tandem mass spectrometry has been developed and used for the separation and determination of 1H-benzotriazole, imidacloprid and clarithromycin in wastewater samples. The analytes were separated on a ZORBAX SB-C3 column with a gradient elution within 3.0 min. The analytes were isolated from wastewater samples by solid-phase extraction. Extraction was carried out on the Oasis HLB cartridges and methanol:acetone (1:1, v/v) was used for elution. The working ranges of the developed method were 1–100 ng L−1. The recovery ranged from 62% to 102% and intra- and inter-day precision was lower than 13% for all analytes. Finally, the optimised method was applied for the determination of the organic micropollutants in wastewater samples: (artificial) raw wastewater, wastewater treated using aerated activated sludge method and wastewater from an Upper Silesia wastewater treatment plant. In addition, transformation products of the analytes in the treated wastewater samples were tentatively identified.

Simultaneous Determination of Amino Acids and Biogenic Amines in Tuna and Mahi-Mahi by Reversed-Phase Ultra-High Performance Liquid Chromatography

ABSTRACTTuna and mahi-mahi are two major fish species responsible for histamine poisoning. This research developed a rapid ultra-high performance liquid chromatography (UHPLC) method to determine amino acids, histamine, and other biogenic amines that can act as co-indicators of histamine poisoning in tuna and mahi-mahi. The modified UHPLC method could simultaneously determine four biogenic amines and 10 major free amino acids in mahi-mahi (Coryphaena hippurus) and yellowfin tuna (Thunnus albacares) within 17.5 min. This UHPLC method showed good linear response, sensitivity, resolution, recovery, repeatability, and number of theoretical plates. The concentrations of detected amino acids, biogenic amines in mahi-mahi and tuna, and their relationships with fish spoilage grade were determined by this UHPLC method. The developed UHPLC method is a rapid and accurate way to monitor quality changes of mahi-mahi and tuna by inspecting the changes of amino acids and biogenic amines.

Determination of Amlodipine Besilate and Azilsartan Medoxomil by UHPLC, HPTLC and Spectrophotometric Techniques

Aims: To develop methods with complete validation according to ICH guidelines and to be applied for the determination of both drugs in laboratory prepared mixtures and in synthetic tablets.
 Study Design: Ultra high performance liquid chromatography (UHPLC), High performance thin layer chromatography (HPTLC) and visible spectrophotometric methods are developed for determination of amlodipine besilate and azilsartan medoxomil in laboratory-prepared mixtures and in synthetic tablets.
 Methodology: Two techniques have been developed for the simultaneous determination of amlodipine besilate and azilsartan medoxomil in pure form and synthetic tablets. The first was UHPLC in which separation was achieved on a C18 column using 0.1% o-phosphoric acid - acetonitrile - methanol (60:10:30, by volume) as mobile phase with detection at 243nm. The second was HPTLC where separation was performed on silica gel 60 F254 plates using chloroform- tolune-methanol-glacial acetic acid (7: 1.5: 1.5: 0.5 by volume) as a developing system and UV detection at 243nm. In addition, visible- spectrophotometric method was developed for determination of amlodipine besilate in presence of azilsartan medoxomil through formation of yellowish orange colored product after reaction of amlodipine besilate with anisaldehde in acid medium with λmax at 443 nm.
 Results: UHPLC method was linear over the concentration ranges of 2-20 μg/ mL and 4-40 μg/ mL while HPTLC method was linear over the concentration ranges of 0.2 -4.0 μg/ spot and 0.5-8.0 μg/ spot for amlodipine besilate and azilsartan medoxomil, respectively. The visible spectrophotometric method was found to be valid over the concentration range of 10–80 μg/mL for amlodipine besilate.
 Conclusion: The proposed three techniques are rapid, accurate and precise, thus can be effectively applied for the routine estimation of both drugs in bulk and in their combined formulations.

Determination of serum imatinib and its' metabolite in patients chronic myeloid leukemia

IntroductionImatinib has favorable pharmacokinetic properties, but primary and secondary resistance mechanisms may cause a decrease in clinical response over time. There is a positive correlation between serum imatinib concentrations and treatment response. Our aim was to develop a method for the measurement of imatinib and its' active metabolite N-desmethyl imatinib. MethodsSerum imatinib and N-desmethyl imatinib levels were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and validation studies were carried out according to CLSI (The Clinical & Laboratory Standards Institute) protocols. Serum samples were collected from 40 patients with chronic myeloid leukemia (CML) and analyzed with LC-MS/MS and ultra high-performance liquid chromatography (UHPLC) methods. ResultsThe linearity range and correlation coefficient were 12.2–12,500 ng/mL and 0.9987 for LC-MS/MS method, respectively. Limit of quantitation was determined as 24.4 ng/mL. The retention times of imatinib and N-desmethyl imatinib were 1.66 and 1.60 min, respectively. There was no statistically significant difference between the results of both methods. DiscussionThis LC-MS/MS method is cost-effective and has adavantages such as using low serum volumes, requiring simple pretreatment steps (only protein precipitation) and reduced turnaround times for analysis.

A UPLC/DAD method for simultaneous determination of empagliflozin and three related substances in spiked human plasma

A simple, rapid and sensitive ultrahigh performance liquid chromatographic method was developed for the determination of the anti-diabetic drug: empagliflozin (EMPA) and three related substances in spiked human plasma, using dapagliflozin (DAPA) as an internal standard and tetrahydrofuran as a plasma protein precipitating agent. The chromatographic separation was achieved on an Acquity “UPLC® BEH” C18 column (50 mm × 2.1 mm i.d, 1.7 µm particle size), and a mobile phase consisting of aqueous trifluoroacetic acid (0.1%, pH 2.5): acetonitrile (60:40, v/v) at a flow rate of 0.5 mL/min. Upon using the UPLC system, the run time could be reduced to less than 1.2 min, and the solvents consumption decreased to 0.36 mL of acetonitrile per run. The response was linear over a concentration range of 50–700 ng/mL and 40–200 ng/mL (r2 = 0.9994–0.9999) with lower limits of detection and quantification (LOD/LOQ) of 15/50, 11.5/40, 12/40 and 12.5/40 ng/mL for EMPA and the three related substances, respectively. Good accuracy was obtained with mean percentage recoveries ≥ 96.97% for the studied compounds. The method was validated according to the ICH guidelines and was found suitable for routine analysis of EMPA and its related substances in human plasma.

Forced degradation of tacrolimus and the development of a UHPLC method for impurities determination.

An ultra-high performance liquid chromatography method for simultaneous determination of tacrolimus impurities in pharmaceutical dosage forms has been developed. Appropriate chromatographic separation was achieved on a BEH C18 column using gradient elution with a total run time of 14 min. The method was applied to analyses of commercial samples and was validated in terms of linearity, precision, accuracy, sensitivity and specificity. It was found to be linear, precise and accurate in the range of 0.05 to 0.6 % of the impurities level in pharmaceutical dosage forms. Stability indicating power of the method was demonstrated by the results of forced degradation studies. The forced degradation study in solution revealed tacrolimus instability under stress alkaline, thermal, light and photolytic conditions and in the presence of a radical initiator or metal ions. The drug was stable at pH 3-5. Solid-state degradation studies conducted on amorphous tacrolimus demonstrated its sensitivity to light, elevated temperature, humidity and oxidation.

A rapid and efficient extraction method based on industrial MCM-41-miniaturized matrix solid-phase dispersion extraction with response surface methodology for simultaneous quantification of six flavonoids in Pollen typhae by ultra-high-performance liquid chromatography.

An industrial MCM-41-miniaturized matrix solid-phase dispersion extraction coupled with response surface methodology was explored to determine L-epicatechin, typhaneoside, isorhamnetin-3-O-neohespeidoside, naringenin, kaempferol, and isorhamnetin in Pollen typhae by ultra-high performance liquid chromatography connected to a photodiode array detection. Several variables were optimized in detail, including mesh number of sieve, type of adsorbent, mass ratio of sample to adsorbent, grinding time, methanol concentration, and elution volume. Central composite design was applied to optimize the best conditions for the maximum yields of the total flavonoids. The results displayed a good linear relationship (R>0.9992) and the recoveries ranged from 92.9 to 103% (RSD<4.53%) of the six flavonoids. The optimal method with high efficiency and low consumption was obviously better than heating reflux and ultrasonic extraction. It was proven that the developed industrial MCM-41-miniaturized matrix solid-phase dispersion extraction coupled with simple ultra-high performance liquid chromatography method could be a rapid and efficient tool for extraction and determination of flavonoids in natural products.

Riboflavin, niacin, folate and vitamin B12 in commercial microalgae powders

This study aimed to investigate the riboflavin, niacin, folate and B12 content in microalgae powders. Riboflavin was determined with an ultra-high-performance liquid chromatographic (UHPLC) method after extraction and a two-enzyme treatment. Niacin analysis involved mild acidic hydrolysis, with niacin quantitated as the sum of nicotinic acid and nicotinamide using a UHPLC method. Both a microbiological (MBA) and a UHPLC method was used for vitamin B12 quantification as a cyanocobalamin (CNCbl) and UHPLC-mass spectrometry was used to confirm the vitamin B12 form. Total folate was determined both with MBA and as a sum of the vitamers with a UHPLC method after extraction and tri-enzyme treatment.The riboflavin and niacin content varied from 21 to 41 μg/g and 0.13–0.28 mg/g, respectively, in Chlorella sp., Spirulina (Arthrospira sp.) and Nannochloropsis gaditana powders. Chlorella powders were, on average, richer in total folate (19.7 μg/g) than Spirulina powders were (3.5 μg/g). The sum of the folate vitamers determined with UHPLC matched better with the microbiological total folate content in Chlorella than in Spirulina powders. Pseudovitamin B12 was the predominant form over active vitamin B12 in Spirulina powders, whereas Chlorella sp. and N. gaditana powders solely contained active vitamin B12 up to 2.1 μg/g.

Determination of epoxide impurity in sarpogrelate hydrochloride intermediate by UHPLC and column-switching liquid chromatography

The determination of genotoxic impurities, which is closely related to toxicological concern and daily dose, plays a key role in drug quality control. Epoxide impurity is a kind of genotoxic impurity with an epoxy ring structure during the synthesis process of sarpogrelate hydrochloride. According to the sarpogrelate hydrochloride daily dose, epoxide impurity is limited to the under 5 ppm level. The liquid chromatographic-tandem mass spectrometric (LC/MS/MS) or the gas chromatography-mass spectrometric (GC/MS) method is commonly used to characterize the epoxide impurity of sarpogrelate hydrochloride intermediates. However, these methods are not simple or economical enough to detect epoxide impurity. In this study, we resolved the problem by using the most common UV method with two ideas: one was to improve the absolute sensitivity, and the other was to reduce matrix effects. Both ultra high-performance liquid chromatography (UHPLC with high sensitivity LightPipe™ flow cells) and column-switching liquid chromatography methods were developed and validated for the quantitative determination of epoxide impurity in sarpogrelate hydrochloride intermediates. The limits of detection (LODs) of the UHPLC and column-switching liquid chromatography methods were 0.09 ppm (0.09 μg/g) and 0.33 ppm (0.33 μg/g), and the recovery rates of both methods were 87.2%–132.1% and 97.4%–100.1%, respectively. Both methods established and provided guidance for analysts to develop procedures for impurity control, especially for structures of impurity with similar matrices.

Bioactivities of serotonin transporter mediate antidepressant effects of Acorus tatarinowii Schott

Ethnopharmacological relevanceAcrorus tatarinowii Schott has been widely used in the treatments of neuropsychiatric and digestive disorders in clinical practices of traditional Chinese medicine for thousands of years. Both clinical and preclinical studies demonstrated antidepressant effects of A. tatarinowii. However, the possible action mechanisms of antidepressant effects of A. tatarinowii remain unraveled.Aim of the study: The present study aimed to investigate the roles of serotonin transporter (SERT) in antidepressant effects of A. tatarinowii. Materials and methodsAntidepressant effects of water extract of A. tatarinowii were evaluated by forced swimming test (FST), tail suspension test (TST) and locomotor activity test. The water extract was analyzed by ultra high performance liquid chromatography (UPLC) method. Two major fractions of A. tatarinowii, petroleum ether extract and water extract after petroleum ether processed, were prepared and analyzed by UPLC method. Further, volatile oil extracted by ether extraction, solid phase micro-extraction (SPME) and hydro-distillation were compared and analyzed by gas chromatography-mass spectrometer (GC-MS) method. Finally, major constituents of water extract of A. tatarinowii were isolated by preparative high performance liquid chromatography (HPLC) and identified by extensive spectroscopic analyses. Effects of all of the above mentioned samples on SERT activity were tested by a high content assay (HCA). ResultsResults of FST, TST and locomotor activity confirmed that water extract of A. tatarinowii significantly decreased mice immobility time but did not change mice locomotor activity. UPLC analysis results revealed that the water extract contained trace amount of β-asarone (0.0004206%) and α-asarone (0.0001918%). HCA results demonstrated that the water extract significantly enhanced SERT activity at 100 μg/mL. Further, GC-MS and UPLC analyses revealed that petroleum ether extract contained high content of β-asarone (45.63%) and α-asarone (12.50%). GC-MS analysis results demonstrated that the volatile oil extracted by ether extraction, SPME and hydro-distillation contained similar major components. HCA results verified that the petroleum ether extract significantly enhanced SERT activity at 1.56 μg/mL. Moreover, UPLC analysis of water extract after petroleum ether processed did not show any characteristic peaks. HCA results demonstrated that this extract significantly inhibited SERT activity at 50–100 μg/mL. Finally, phytochemistry investigation on the water extract of A. tatarinowii afforded seven constituents including veratric acid (9), anisic acid (7), 3,4,5-trimethoxybenzoic acid (3), trans-isoferulic acid (2), 2,4,5-trimethoxybenzoic acid (11), 4-hydroxybenzoic acid (6) and syringic acid (13). Their structures were established on the basis of nuclear magnetic resonance (NMR) and mass spectrometer (MS) data and comparative UPLC analyses. HCA results demonstrated the major components of the water extract of A. tatarinowii demonstrated SERT enhancement/inhibition activities. ConclusionsThis study first systematically demonstrated the roles of SERT activity in antidepressant effects of A. tatarinowii, including water extract, major fractions and main constituents. These results revealed that A. tatarinowii could regulate SERT activities in bidirectional ways.

Validated Stability Indicating HPTLC, UHPLC and UV-Spectrophotometric Techniques for the Determination of Bepotastine Besilate in Presence of Its Oxidative Degradate

The study is aimed at developing methods which have a complete validation as stipulated in the ICH guidelines and to be applied for the determination of Bepotastine besilate (BB) in pure form and in pharmaceutical formulations in the presence of its oxidative degradation product. High performance thin layer chromatography (HPTLC), Ultra high performance liquid chromatography (UHPLC) and different spectrophotometric methods (first derivative, first derivative of ratio spectra and ratio difference are developed for simultaneous determination of bepotastine besilate in laboratory-prepared mixtures of bepotastine besilate with its oxidative degradate and in pharmaceutical formulations were used in the study design. Firstly, HPTLC was performed and separation occurred on silica gel 60 F254 plates, with butanol: ammonia (8:2, v/v) as a developing system. UHPLC in which separation occurred on a Kinetex C 18 column using methanol- 0.1% O-phosphoric acid - acetonitrile (70:20:10, by volume) as mobile phase, followed. And lastly was UV/Vis spectrophotometry which included first derivative determination of the drug at 252.6 nm, first derivative of ratio of peak amplitudes at 233.4, 250 and 275.6 nm and the ratio difference with the amplitude difference between (240 nm and 260 nm). Result showed that HPTLC method was applicable over the concentration range of 0.5-5 μg / band, while UHPLC method was linear over the concentration 2- 12 μg/mL and spectrophotometric methods were linear over the concentration range 20-120 μg/mL for bepotastine besilate. The proposed three techniques are quite accurate and precise. They can be used for routine analysis of bepotastine besilate in pharmaceutical formulation and stability indicating methods.

INTERNATIONAL CONFERENCE ON HARMONIZATION RECOMMENDED FORCED DEGRADATION STUDIES AND DEVELOPMENT OF A NEW VALIDATED ISOCRATIC REVERSE-PHASE ULTRA HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR THE SIMULTANEOUS ESTIMATION OF TELMISARTAN AND AMLODIPINE I

Objective: The objective of this research work is to develop a simple, rapid, cost-effective, accurate, precise, and robust stability-indicating reversephase ultra-high performance liquid chromatography method for the quantitative estimation of telmisartan (TEL) and amlodipine (AMD) in combined tablet dosage form.&#x0D; Methods: The method uses Poroshell 120EC-C18 column (4.6 mm × 50 mm, 2.7 μm) with an optimized mobile phase containing acetonitrile:50 mM ammonium acetate buffer in the ratio (45:55 v/v), pH adjusted to 4.5 with acetic acid. The flow rate was 0.5 ml/min, column temperature at 25°C and detection was monitored by a PDA detector at a wavelength of 245 nm. International Conference on Harmonization (ICH) recommended stress degradation studies were performed on TEL, AMD standard drugs, and tablet formulations; further stressed samples were analyzed by the proposed method.&#x0D; Results: Major degradation of TEL and AMD was observed under acidic, alkali, hydrolytic and oxidation conditions. The described method was validated as per the ICH guideline and validation parameters such as system suitability, linearity, accuracy, precision, specificity, and robustness results were within acceptable limits.&#x0D; Conclusion: The method was found to be suitable and accurate for quantitative estimation and stability study of title drugs in pharmaceutical preparations.

UHPLC Method Development for Determining Sitagliptin and Dapagliflozin in Lipid-Based Self-Nanoemulsifying Systems as Combined Dose in Commercial Products and its Application to Pharmacokinetic Study of Dapagliflozin in Rats

Anew simple, precise, selective and reproducible reversed-phase ultra-high performance liquid chromatography (RP-UHPLC) method has been developed and validated for the first time in simultaneous determination of two anti-diabetic drugs, sitagliptin (SN) and dapagliflozin (DN), in lipid-based self-nanoemulsifying formulations as a new combined preparation dose. The chromatographic separation was carried out on a reversed-phase Acquity® BEH C18 column using methanol–acetonitrile–water mixture at 24/18/58 (%v/v/v) ratio as the mobile phase. The isocratic flow rate was 0.4 mL/min with a run time of 6 min. The UV detection of both SN and DN was performed at 210 nm. The method was validated over a concentration range of 100 – 10000 ng/mL (r2 = 0.9997 and 0.9999 for SN and DN, respectively). The selectivity, specificity, recovery, accuracy and precision for determining SN and DN in lipid-based formulation were validated. The lower limits of detection and quantitation of the method were 27 and 89 ng mL–1 for SN and 19 and 61 ng mL–1 for DN, respectively. The intra- and inter-day coefficients of variation were less than 4%. The validated method has been successfully applied to quantify both SN and DN in self-nanoemulsifying formulation of combined dosage form. In addition, the proposed method was also applied for in vivo pharmacokinetic study using an animal model (rat) to further illustrate the scope and application of the proposed method.

Development of a high resolution mass spectrometry method for the determination of danshensu and salvianolic acid B

In this work, a highly sensitive ultrahigh-performance liquid chromatography and quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) method has been developed and validated by the quantifications of danshensu (DSS) and salvianolic acid B (SAB) in Danhong injection (DHI) in human plasma, urine and feces for the pharmacokinetic and excretion studies. The DSS and SAB were extracted from biological matrix by direct protein precipitation with fivefold volume of methanol and separated by a Waters CORTECS C18+ chromatographic column (100 × 2.1 mm, 2.7 μm) with gradient elution (mobile phase: methanol–H2O, both containing 0.1% formic acid). The detection of analytes was achieved using negative ion ESI in Full MS/dd-MS2 (data-dependent MS2) scan mode. The human subjects received either a single intravenous injection (i.v.) dose (containing 0.331 mg/kg DSS and 0.117 mg/kg SAB) or a single intramuscular injection (i.m.) dose (containing 0.663 mg/kg DSS and 0.234 mg/kg SAB) of DHI. For i.v., the Cmax, half-life (t1/2) and AUC0−∞ were 906.2 ± 31.82 μg/L, 0.604 ± 0.103 h and 242.19 ± 18.24 μg/L × h for DSS and 1118.5 ± 35.64 μg/L, 0.893 ± 0.159 h and 277.63 ± 11.24 μg/L × h for SAB, respectively. For i.m., the Cmax, Tmax, t1/2 and AUC0−∞ were 243.0 ± 29.1 μg/L, 0.67 h, 0.71 ± 0.03 h and 318.98 ± 7.62 μg/L × h for DSS and 119.4 ± 6.51 μg/L, 0.50 h, 1.08 ± 0.18 h and 185.63 ± 4.23 μg/L × h for SAB, respectively. Based on the AUC0−∞ obtained from i.m. and i.v. administrations, the absolute bioavailabilities (F) were estimated to be 65.9% for DSS and 33.4% for SAB.

Development and Validation of a UHPLC-DAD Method for the Quantitative Analysis of Major Dihydrochalcone Glucosides from Thonningia sanguinea VAHL.

Thonnigia sanguinea is a plant widely used in traditional African medicine against a variety of diseases. The obligate parasite is growing throughout tropical African forests and utilizes a large variety of hosts. Dihydrochalcone glucoside derivatives isolated from the subaerial parts of this plant were identified as potential antidiabetic lead compounds. In this study, an ultrahigh-performance liquid chromatographic method coupled with a photodiode array detector was developed for the quantitation of six major dihydrochalcone derivatives. The analytes were baseline separated in complex samples within 14 minutes on a Phenomenex Luna Omega 1.6 µm C18 column using a mobile phase consisting of water and acetonitrile (each + 0.01% trifluoroacetic acid) in gradient elution. Method validation confirmed the selectivity, linearity (R2 ≥ 0.9992), precision (inter-day ≤ 1.98%, intraday ≤ 2.00%), and accuracy (recovery rates of 97.4 - 106.3% for all analytes). At 280 nm, the LODs and LOQs were found to be lower than 1.42 and 4.30 µg/mL, respectively. Eight plant batches from the northern Angolan province of Uíge (collected in the wild or bought on markets) were extracted with methanol using an ultrasound-assisted extraction protocol and subsequently analyzed with the validated method. Results indicated high contents of dihydrochalcone glucosides in all eight samples. Most notably, the two bioactive constituents thonningianin A and B were present in fairly large amounts (2.42 - 5.35 w%).

The content of selected vitamins and iodine in mare’s milk

The aim of the study was to contribute to scarce literature data on the content of selected vitamins and iodine in mare’s milk. The study focused on the content of selected lipophilic vitamins (A, E), hydrophilic vitamins B1, B2, B6) and iodine in milk samples obtained from 8 mares during 6 months of lactation. The content of micronutrients was evaluated according to the stage of lactation. Vitamins B2 and B6 were determined using ion-pair reversed phase high performance liquid chromatography with fluorescence detection, vitamin B1 by fluorescence detection by reversed-phase liquid chromatography, vitamins A and E by the ultra-high performance liquid chromatography method with ultraviolet and fluorescence detection and iodine by the spectrophotometric method. Mean concentrations of vitamins A (0.06 ± 0.08 mg/l), E (0.083 ± 0.14 mg/l), B1 (256.24 ± 44.19 µg/l), B2 (111.40 ± 81.88 µg/l), B6 (0.30 ± 0.12 mg/l) reached lower values in mare’s milk compared to cow’s milk. The mean value of iodine in mare’s milk reached 44.48 ± 54.45 µg/l. Lactation stage proved to be a significant factor for vitamin B6 and iodine.

UHPLC analysis of major functional components in six types of Chinese teas: Constituent profile and origin consideration

In this study, an effective ultra-high performance liquid chromatography (UHPLC) method for simultaneous determination of 15 constituents of catechins, caffeine, gallic acid and theaflavins was developed. Employing the developed UHPLC method, the contents of the 15 constituents were analyzed in 245 samples of 6 types of Chinese teas collected from different places. The catechin contents in these samples ranged from 16.50 to 94.35 mg/g. Theaflavins were not detected in green tea and white tea, and caffeine did not strongly relate to germplasm resource or fermentation degree. Principal component analysis (PCA) of UHPLC data revealed that white, oolong and green tea samples could be clustered closely, demonstrating notable difference of functional components between the three types of teas resulting from preparation process and varieties. The constituent profile could be primarily attributed to the structural transformation of catechins and pigments during tea processing. This study would advance our understanding of chemical profile of Chinese teas afforded by different processing technologies.