HPTLC–densitometric and HPTLC–MS methods for analysis of flavonoids

HPTLC silica gel plates without and with fluorescence indicator F₂₅₄ in combination with n-hexane–ethyl acetate–formic acid (20:19:1, v/v/v) as a developing solvent were explored for the HPTLC–densitometric and HPTLC–MS/(MSⁿ) analyses of flavonoids. Pre-development of the plates with chloroform–methanol (1:1, v/v) was needed for reliable HPTLC–densitometric analyses of flavonoid aglycones in the whole R_F range, while 2-step pre-development (1^st methanol–formic acid (10:1, v/v), 2^nd methanol), that decreased background signals of formic acid adducts, was required for HPTLC–MS analyses. Optimization with conditioning of the adsorbent layer with water before development and saturation of the twin trough chamber resulted in required decrease of the R_F values of studied flavonoids (flavone, apigenin, luteolin, chrysin, quercetin dihydrate, myricetin, kaempferide, kaempferol, naringenin, pinocembrin).

Detection was performed based on fluorescence quenching (on the plates with F₂₅₄), natural fluorescence and after post-chromatographic derivatization with natural product reagent without or with further enhancement and stabilization of fluorescent zones with polyethylene glycol (PEG 400 or PEG 4000) or paraffin–n-hexane reagents. For all three reagents, drying temperature and time passed after drying influenced the intensity, which was increasing the first 20?min, and the stability (less than 2?h for PEGs and at least 24?h for paraffin–n-hexane) of the standards’ zones.

Optimal wavelengths for densitometric evaluation were selected based on in-situ absorption spectra scanned before and after derivatization and after stabilization. The developed method was tested via analyses of propolis, roasted coffee, rose hip, hibiscus, rosemary and sage crude extracts. To further increase the reliability of the obtained densitometric results HPTLC–MS/(MSⁿ) analyses of all crude extracts were performed. Several phenolic and non-phenolic compounds were tentatively identified.

Some possible interferences with phenolic acids (chlorogenic acid, rosmarinic acid, protocatechuic acid, gallic acid, syringic acid, ellagic acid, trans-cinnamic acid, o-coumaric acid, m-coumaric acid, p-coumaric acid, caffeic acid, ferulic acid, sinapic acid) that are often present in the extracts together with flavonoids were also examined.     

Development and validation of a simple HPTLC method for the determination of new hepatitis C subtype 4 antiviral agents in their tablet dosage form

A new, simple, precise, accurate and selective high-performance thin-layer chromatographic (HPTLC) method has been developed and validated for the simultaneous determination of ledipasvir and sofosbuvir in their tablet dosage form. Chromatographic separation was carried out on Merck TLC aluminum sheets of silica gel 60 F₂₅₄ using ethyl acetate:hexane:methanol in the ratio of 8:1.25:0.75 (% v/v) as the mobile phase followed by densitometric measurement at 256 nm. The method was validated in terms of linearity, accuracy, precision, limit of detection, limit of quantification and specificity in accordance with the International Conference on Harmonization (ICH) guidelines. The calibration curve was found to be linear between 60 to 1980 and 45 to 3600 ng/band for ledipasvir and sofosbuvir, respectively, with significantly high value of regression coefficient (r² > 0.9999) with linear and homoscedastic residuals. The limits of detection and quantification were found to be 16.5 and 50 ng/band, respectively, for ledipasvir and 13 and 39.5 ng/band, respectively, for sofosbuvir. Comparative study was performed between the developed HPTLC method and the reported high-performance liquid chromatography (HPLC) method. The quantitative results of the two analytical methods did not show statistically significant difference, whereas the developed HPTLC method is both time- and cost-effective.

     

Cloud-point extraction and reversed-phase high-performance liquid chromatography for the determination of carbamate insecticide residues in fruits

A cloud-point extraction (CPE) method using Triton X-114 non-ionic surfactant was developed for the extraction and preconcentration of carbamate insecticide residues (i.e., methomyl, propoxur, carbofuran, carbaryl, isoprocarb, and promecarb) in fruit samples. The optimum conditions of CPE were 1.5% (w/v) Triton X-114, 7.0% (w/v) NaCl and 20 min equilibrated at 45 °C. The surfactant-rich phase was then analyzed by reversed-phase high-performance liquid chromatography with ultraviolet detection at 270 nm, under gradient separation using methanol and 0.1% (v/v) acetic acid. Under the study conditions, six carbamate insecticides were successfully separated within 27 min. Good reproducibility was obtained with the relative standard deviation of <3% for retention time and <9% for peak area. Limits of detection in the studied fruit samples were in the range of 0.1–1.0 mg kg⁻¹. No carbamate insecticides were detected in the studied fruit samples. The high recoveries of the spiked fruit samples were obtained in the range 80.0–107%. The CPE method has been shown to be a potential useful methodology for the preconcentration of the target analytes, with a preconcentration factor of 14. Moreover, the method is simple, has high sensitivity, consumes much less solvent than traditional methods, and is environmental friendly.     

Comparative study of HPLC-DAD and HPTLC for the simultaneous determination of a new multitarget antidiabetic ternary mixture in combined tablets

Qternmet XR® (FDA approval, May 2019) is a multitarget anti-diabetic drug combination composed of metformin (MET), saxagliptin (SAX) and dapagliflozin (DAP). To our present knowledge, no analytical reports were found in the scientific databases for the simultaneous quantification of MET, SAX and DAP in their ternary combined tablets, moreover, no articles have attempted the simultaneous estimation of the cited drugs in any matrix using high-performance liquid chromatography with diode-array detection (HPLC–DAD) or high-performance thin-layer chromatography (HPTLC) technique. The current work represents a comparative study on two developed and validated chromatographic methods for the simultaneous determination of the ternary mixture (MET, SAX and DAP) in pure form and in combined tablet dosage form. The first method is reversed-phase HPLC using Agilent C18 column (4.6 × 250 mm, 5 μm p.s.) with a mobile phase consisting of acetonitrile and acidic aqueous phase pH 3 with a photodiode array detection at 230 nm. The second method is HPTLC in which drug solutions were applied to Merck HPTLC silica gel plates developed with a mixture of chloroform:methanol:water:acetic acid (7.4:2.6:0.5:0.01, v/v) and scanned at 224 nm. Both methods were fully validated following the ICH guidelines in terms of linearity, accuracy, precision, selectivity and robustness.

Representative HPLC (a) and HPTLC (b) chromatograms for a ternary mixture of metformin (MET), saxagliptin (SAX) and dapagliflozin (DAP)

     

Rapid and cost‐effective method for the simultaneous quantification of seven alkaloids in Corydalis decumbens by microwave‐assisted extraction and capillary electrophoresis

A rapid and cost‐effective method based on microwave‐assisted extraction followed by capillary electrophoresis was developed for simultaneous quantification of seven alkaloids in Corydalis decumbens for the first time. The main parameters affecting microwave‐assisted extraction and capillary electrophoresis separation were investigated and optimized. The optimal microwave‐assisted extraction was performed at 40°C for 5 min using methanol/water (90:10, v/v) as the extracting solvent. Electrophoretic separation was achieved within 15 min using an uncoated fused‐silica capillary (50 μm internal diameter and 27.7 cm effective length) and a 500 mM Tris buffer containing 45% v/v methanol (titrated to pH^* 2.86 with H₃PO₄). The developed method was successfully applied to the quantification of seven alkaloids in Corydalis decumbens obtained from different regions of China. The combination of microwave‐assisted extraction with capillary electrophoresis was an effective method for the rapid analysis of the alkaloids in Corydalis decumbens .     

Stability Indicating HPTLC and LC Determination of Dasatinib in Pharmaceutical Dosage Form

Two sensitive and reproducible methods are described for the quantitative determination of dasatinib in the presence of its degradation products. The first method was based on high performance thin layer chromatography (HPTLC) followed by densitometric measurements of their spots at 280 nm. The separation was on HPTLC aluminium sheets of silica gel 60 F₂₅₄ using toluene:chloroform (7.0:3.0, v/v). This system was found to give compact spots for dasatinib after development (R _F value of 0.23 ± 0.02). The second method was based on high performance liquid chromatography (HPLC) of the drug from its degradation products on reversed phase, PerfectSil column [C₁₈ (5 μm, 25 cm × 4.6 mm, i.d.)] at ambient temperature using mobile phase consisting of methanol:20 mM ammonium acetate with acetic acid (45:55, v/v) pH 3.0 and retention time (t _R = 8.23 ± 0.02 min). Both separation methods were validated as per the ICH guidelines. No chromatographic interference from the tablet excipients was found. Dasatinib was subjected to acid–alkali hydrolysis, oxidation, dry heat, wet heat and photo-degradation. The drug was susceptible to acid–alkali hydrolysis and oxidation. The drug was found to be stable in neutral, wet heat, dry heat and photo-degradation conditions. As the proposed analytical methods could effectively separate the drug from its degradation products, they can be employed as stability indicating.     

LC Method for Quantification of ent-11α-Hydroxy-15-oxo-kaur-16-en-19-oic Acid in Rabbit Plasma: Validation and Application to a Pharmacokinetic Study

ent-11α-Hydroxy-15-oxo-kaur-16-en-19-oic acid (5F), a diterpenoid isolated from the Chinese herb Pteris semipinnata L, has been suggested to show antitumor properties. A simple and sensitive LC method was developed for the determination of 5F in rabbit plasma. The method involved liquid–liquid extraction using ethyl acetate under acidic conditions using naproxen as an internal standard. Separations were performed on a reversed-phase column with a mixture of 1% (v/v) glacial acetic acid and methanol (45:55, v/v) as mobile phase and UV detection was utilized at 242 nm. The calibration plot was linear in the range 0.20–10.0 μg mL⁻¹ (correlation coefficients r ² > 0.998). The detection limit was 0.20 μg mL⁻¹, mean extraction recovery was above 82%, intra-day precision of the method was less than 6.4%, and inter-day precision was better than 8.7%, respectively. The validated assay was found to be suitable for the pharmacokinetic study of 5F in rabbits.

     

Application of dried blood spots combined with high-performance liquid chromatography coupled with electrospray ionisation tandem mass spectrometry for simultaneous quantification of vincristine and actinomycin-D

A sensitive, specific and efficient high-performance liquid chromatography-tandem mass spectrometry assay for the simultaneous determination of vincristine and actinomycin-D in human dried blood spots is presented. Dried blood spots were punched out of a collection paper with a 0.25-in.-diameter punch. The analytes were extracted from the punched-out disc using sonication during 15 min in a mixture of acetonitrile–methanol–water (1:1:1, v/v/v) containing the internal standard vinorelbine. Twenty-microlitre volumes were injected onto the HPLC system. Separation was achieved on a 50 × 2.1 mm ID Xbridge C₁₈ column using elution with 1 mM ammonium acetate–acetonitrile (70:30, v/v) adjusted to pH 10.5 with ammonia and run in a gradient with methanol at a flow rate of 0.4 mL/min. HPLC run time was 6 min. The assay quantifies vincristine from 1 to 100 ng/mL and actinomycine-D from 2 to 250 ng/mL using a blood sample obtained by a simple finger prick. Validation results demonstrate that vincristine and actinomycin-D can be accurately and precisely quantified in human dried blood spots with the presented method. The assay can now be used to support clinical pharmacologic studies with vincristine and actinomycin-D.     

Determination of Abamectin Residues in Avocados by Microwave-Assisted Extraction and HPLC with Fluorescence Detection

In this article a new analytical method for the confirmation and quantification of abamectin residues in avocados is described. The method allows a fast analysis of abamectin homologues using microwave assisted extraction (MAE), solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with fluorescence (FL) detection using trifluoroacetic anhydride (TFAA) and N-methylimidazole (NMIM) as derivatizing agents. The mobile phase consisted of water, methanol and acetonitrile (5:47.5:47.5 v/v/v) and was pumped at a rate of 1.1 mL min⁻¹ (isocratic elution). Homogenized avocado samples were extracted once with 20 mL acetonitrile:water 4:1 (v/v) in a microwave oven for 26 min at 700 W with a maximum temperature of 80 °C. MAE operational parameters were optimized by means of an experimental design. Extracts were cleaned using C₁₈ SPE cartridges. Average recoveries of the method at four spiked levels (0.005, 0.01, 0.10 and 1.0 mg kg⁻¹) were found to be in the range 90–100% with good precision (RSD < 12%). The limits of detection (LODs) and quantification (LOQs) of the whole method were 0.001 and 0.003 mg kg⁻¹, respectively, which are lower than the maximum residue limit (MRL) established by the Spanish and the European legislation in avocados (0.01 mg kg⁻¹). Several avocado samples previously treated with the pesticide were also analyzed.     

Simultaneous determination of theophylline,tolbutamide, mephenytoin,debrisoquin, and dapsone in human plasma using high‐speed gradient liquid chromatography/tandem mass spectrometry on a silica‐based monolithic column

Theophylline, tolbutamide, mephenytoin, debrisoquin, and dapsone are marker substrates for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, respectively. A silica‐based monolithic column (Chromolith SpeedROD RP‐18e, 50×4.6 mm) was used to separate these five marker substrates of cytochrome P450 within only 84 s. Linear gradient elution was from acetonitrile‐water‐formic acid (10 : 90 : 1, v/v/v) to acetonitrile‐water‐formic acid (90 : 10 : 1, v/v/v) in 1.4 min. The flow rate was 2.5 mL/min. The retention time was 0.52 min for theophylline, 0.67 min for debrisoquin, 0.78 min for dapsone, 0.96 min for mephenytoin, and 1.13 min for tolbutamide. Detection was by tandem mass spectrometry using a PE Sciex API 3000 mass spectrometer with a Turbo‐Ionspray source in positive mode. A simple protein precipitation method was used. This method was validated over the concentration range of 5–2000 ng/mL based on the sample volume of 0.1 mL.     

A rapid and highly sensitive stability-indicating high-performance thin-layer chromatography technique for the determination of tedizolid phosphate with a classical univariate calibration

No high-performance thin-layer chromatography (HPTLC) techniques have been established for the determination of tedizolid phosphate (TDZP) in pharmaceutical products or physiological fluids. Therefore, a rapid and highly sensitive stability-indicating HPTLC technique has been developed for the determination of TDZP in commercial formulations with a classical univariate calibration. The HPTLC‒densitometry analysis of TDZP was carried out via chloroform‒methanol (90:10, V/V) mobile phase. The determination of TDZP was performed at the wavelength of 300 nm. The proposed HPTLC technique was linear in the range of 10‒2000 ng band^‒1. In addition, the method was found to be highly accurate (% recovery = 98.53‒101.74%), precise (%CV = 0.67‒0.91%), robust (%CV = 0.83‒0.86%), highly sensitive (LOD = 3.41 ng band^‒1, LOQ = 10.23 ng band^‒1) for the determination of TDZP. The proposed technique was also able to detect TDZP in the presence of its degradation products under various stress conditions and it can be considered as a stability-indicating method. The proposed HPTLC technique was applied for the analysis of TDZP in its commercial formulations. The TDZP contents of commercial tablets and injection were determined as 98.41% and 101.23%, respectively. These results suggested that the proposed HPTLC technique can be applied for the routine analysis of TDZP in its commercial products and newly established formulations.

     

LC Method for Determination of Ginkgolic Acids in Mice Plasma and Its Application to a Pharmacokinetic Study

A sensitive and simple LC method for the quantification of ginkgolic acids in mice plasma has been developed. Following acetonitrile deproteinization, samples were separated on a SinoChrom ODS-AP C₁₈ column. The mobile phase was 3% (v/v) acetic acid water solution–methanol (8:92, v/v) at a flow rate of 1.0 mL min⁻¹. Detection was at 310 nm. Calibration curve was linear over the range of 0.25–50 μg mL⁻¹ with intra- and inter-day precisions (RSD%) of less than 9.5%. The extraction recovery ranged from 87.0 to 90.2% (RSD 2.4–6.4%) for ginkgolic acids. The method was successfully applied to the pharmacokinetic study of ginkgolic acids in mice after oral dosing of 1.0 g kg⁻¹.

     

Determination of Flavonoid Markers in Honey with SPE and LC using Experimental Design

Determination of flavonoid markers quercetin, hesperetin, and chrysin, found in north Iranian citrus honey samples, was carried out by solid phase extraction (SPE) and isocratic liquid chromatographic separation using central composite design. Optimum conditions for SPE were achieved using 10 mL methanol/water (13:87, v/v, pH = 7) as the washing solvent and 4 mL methanol for elution. Good clean-up and high recovery >90% were observed for all analytes. The use of water/ACN/THF/AcOH (54:36:5:5, v/v) was found to serve as the optimum mobile phase composition and allowed for the separation of analytes from endogenous compounds present in honey. SPE parameters, such as maximum loading capacity and breakthrough volume, were also determined for each analyte. Limit of detection, linear range, recovery, repeatability of retention times, and peak heights were 3.11 × 10⁻⁸–4.44 × 10⁻⁸ g g⁻¹, 0.50–50.0 μg mL⁻¹ (R ² > 0.99), 90.7–96.9%, 3.0–3.6%, and 1.0–2.6%, respectively. Precision of the overall analytical procedure, estimated by five replicate measurements for quercetin, hesperetin and chrysin in citrus honey, as well as the relative standard deviations were 4.3%, 3.8%, and 5.5%, respectively.

     

A validated quantification of triclosan in toothpaste using high-performance thin-layer chromatography and a 48-bit flatbed scanner

We present a densitometric quantification method for triclosan in toothpaste, separated by high-performance thin-layer chromatography (HPTLC) and using a 48-bit flatbed scanner as the detection system. The sample was band-wise applied to HPTLC plates (10 × 20 cm), with fluorescent dye, Merck, Germany (1.05554). The plates were developed in a vertical developing chamber with 20 min of chamber saturation over 70 mm, using n-heptane–methyl tert-butyl ether–acetic acid (92:8:0.1, V/V) as solvent. The R_F value of triclosan is hR_F = 22.4, and quantification is based on direct measurements using an inexpensive 48-bit flatbed scanner for color measurements (in red, green, and blue) after plate staining with 2,6-dichloroquinone-4-chloroimide (Gibbs' reagent). Evaluation of the red channel makes the measurements of triclosan very specific. For linearization, an extended Kubelka–Munk expression was used for data transformation. The range of linearity covers more than two orders of magnitude and is between 91 and 1000 ng. The separation method is inexpensive, fast and reliable.

     

Nonaqueous CE for Rapid and Sensitive Determination of Matrine and Oxymatrine in Sophora flavescens and Its Medicinal Preparations

A simple, rapid, and sensitive non-aqueous capillary electrophoresis procedure for the quantitative determination of matrine and oxymatrine is established. Optimum separation conditions were obtained when the sample was injected under pressure for 3 s at 50 mbar and separated with the buffer containing 70 mM ammonium acetate, 7.0% (v/v) acetic acid, and 10% (v/v) acetonitrile in methanol medium at 25 kV applied voltage. The analytes were detected at 205 nm. The two alkaloids can be separated within 12 min and quantified with high sensitivity. The method was validated in terms of reproducibility, linearity, and accuracy when applied to the analysis of matrine and oxymatrine in Sophora flavescens and its medicinal preparations.     

Solid-phase micro-extraction procedure for the determination of 1,3-dichloro-2-propanol in water by on-fibre derivatisation with bis(trimethylsilyl)trifluoroacetamide

The headspace solid-phase micro-extraction technique with on-fibre derivatisation followed by gas chromatography-tandem mass spectrometry has been evaluated for the analysis of 1,3-dichloro-2-propanol in water. An asymmetric factorial design has been performed to study the influence of five experimental factors: extraction time and temperature, derivatisation time and temperature and pH. The best extraction performance is achieved in the headspace mode, with 5 mL stirred water samples (pH 4) containing 1.3 g of NaCl, equilibrated for 30 min at 25 °C, using divinylbenzene-carboxen-polydimethylsiloxane as the fibre coating. On-fibre derivatisation has been used for the first time with 50 μL of bis(trimethylsilyl)trifluoroacetamide at 25 °C during 15 min, leading to effective yields. The proposed method provides high sensitivity, good linearity and repeatability (relative standard deviation of 5.1% for 10 ng mL⁻¹ and n = 5). The limits of detection and quantification were 0.4 and 1.4 ng mL⁻¹, respectively. Analytical recoveries obtained for different water samples were approx. 100%.     

Simultaneous Determination of Tinidazole and Furazolidone in Suspension by HPTLC and HPLC

Abstract

High performance thin layer chromatographic (HPTLC) and high performance liquid chromatographic (HPLC) methods were developed for the simultaneous determination of Tinidazole and Furazolidone in suspension.

In the HPTLC method the separation of Tinidazole and Furazolidone was carried out on silica gel 60F₂₅₄ HPTLC glass plate using chloroform:methanol:ammonia (9:1:0.1 v/v) as a mobile phase. Rf values obtained were 0.63 and 0.79 for Furazolidone and Tinidazole respectively. Densitometric evaluation was done at 335 nm. Linearity was obtained within the concentration range 10–50 μg/ml and 3.5–17.5 μg/ml for Tinidazole and Furazolidone respectively.

The second method is based on high performance liquid chromatography on a reversed phase column (μ Bondapak C₁₈) using a mobile phase comprised of water: acetonitrile: triethylamine (80:20:0.1 v/v) adjusted to pH = 3.0 with dil. phosphoric acid. Retention times were 5.24 and 7.82 min for Tinidazole and Furazolidone respectively at a flow rate of 1.5 ml/min. Detection was done at 335 nm. Linearity was obtained within the concentration range 30–180 μg/ml and 10.5–63 μg/ml for Tinidazole and Furazolidone resp.     

Quantitative determination of canagliflozin in human plasma samples using a validated HPTLC method and its application to a pharmacokinetic study in rats

Canagliflozin (CNZ) is the first sodium–glucose co-transporter-2 inhibitor approved for treatment of type 2 diabetes mellitus. In the proposed work, a sensitive, rapid and validated high-performance thin-layer chromatography (HPTLC) method was established for the estimation of CNZ in human plasma for the first time. HPTLC analysis of CNZ and internal standard (sildenafil) was performed on glass coated silica gel 60 F₂₅₄ HPTLC plates using a binary mixture of chloroform–methanol 9:1 (%, v/v) as the mobile phase. Densitometric detection was done at 295 nm. Retardation factor values were obtained as 0.22 and 0.52 for the CNZ and the IS, respectively. The linearity range of CNZ was obtained as 200–3,200 ng/ml. A simple protein precipitation method was used for the extraction of analyte from plasma using methanol. The proposed HPTLC technique was validated for linearity, accuracy, precision and robustness. The proposed HPTLC technique was successfully utilized for the assessment of pharmacokinetic profile of CNZ in rats after oral administration. After oral administration, the peak plasma concentration of CNZ was obtained as 1458.01 ng/ml in 2 h. The proposed HPTLC method could be applied to the study of the pharmacokinetic profile of pharmaceutical formulations containing CNZ.     

LC Separation of Fatty Acid Ceramides Using a Two Column System

Type II ceramides were separated according to the length of their fatty acid alkyl chain using two column system. The packing of the first column was non-polar adsorbent prepared by coating of macroporous spherical silica with cationized poly(vinyl alcohol) of low substitution degree. Commercial normal phase column Lichrosorb Si 60 was used as a second column in this system. Elution was performed in an isocratic mode using methanol:chloroform 50:50 (v/v) as an eluent.

     

Direct separation and quantitative determination of glimepiride isomers by high performance liquid chromatography

A simple and sensitive high‐performance liquid chromatographic procedure for the determination of the trans isomer of glimepiride is reported. Chromatography accomplished direct separation of the cis and trans isomers of glimepiride on a Dikmonsil C18 (250×4.6 mm, 5 μm) column with a mobile phase consisting of methanol‐acetonitrile‐NH₄Ac buffer solution (1.5 mol L^–1, pH = 4.5) (1.1 : 1.3 : 1.0, v/v) at a flow rate 0.5 mL min^–1. The resolution (R_S) was 1.73 with a retention time of 24.885 and 23.018 min for the cis and the trans isomer, respectively. A standard linear calibration curve was established for the trans isomer of glimepiride over the range of 4.95–198.00 μg mL^–1 with a correlation coefficient of 0.99997. This method has been successfully used to analyze four different kinds of glimepiride product.