Quantification of structurally related aliphatic amino alcohols in l‐valinol by hydrophilic interaction liquid chromatography separation combined with postcolumn derivatization and fluorescence detection

The amino alcohols in l‐ valinol were effectively separated and quantified using hydrophilic interaction chromatography with fluorescence detection. The influence of the mobile phase (salt type, buffer concentration, and pH) on retention was studied. A column TSKgel amide and mobile phase consisting of 10 mM acetate buffer pH 4.0 and acetonitrile (20:80, v/v) provided well‐ separated symmetric peaks of analytes. Fluorescence detection was performed using postcolumn derivatization with o‐phtaldialdehyde/2‐mercaptoethanol at an excitation and emission wavelength of 345 and 450 nm, respectively. Simple sample pretreatment and very high sensitivity represent the main advantages of the developed method. After validation, the method was successfully applied to the analysis of commercial samples of l‐ valinol.     

Determination of rimantadine in human urine by HPLC using a monolithic stationary phase and on‐line post‐column derivatization

In the present study, we propose the first HPLC method coupled to postcolumn derivatization for the determination of rimantadine in human urine samples. The analyte and amantadine (internal standard) were isocratically separated using an RP monolithic stationary phase (100 × 4.6 mm id) with a mobile phase consisting of CH₃OH/phosphate buffer (25 mmol/L, pH 3.0) at a volume ratio of 50:50. Postcolumn derivatization involved on‐line reaction with o‐phthalaldehyde (20 mmol/L) and N‐acetyl‐cysteine (5 mmol/L) at alkaline medium (100 mmol/L borate pH 11.0). Spectrofluorimetric detection at λ_ex/λ_em = 340/455 nm enabled the selective and sensitive determination of rimantadine in urine samples at a range of 50–500 ng/mL with an LOD of 5 ng/mL. Human urine samples were analyzed successfully after SPE using hydrophilic‐lipophilic balanced RP cartridges (30 mg/mL, Oasis HLB). Recoveries ranged between 89.7 and 102.7%.     

Development of a novel,fast, simple,nonderivative HPLC method with direct UV measurement for quantification of memantine hydrochloride in tablets

Fast, simple, accurate, and reproducible reverse phase‐high‐performance liquid chromatography method with direct ultraviolet measurement of memantine hydrochloride in tablets was developed, without any chemical derivatization pretreatment. Three main problems appear during chromatographic analysis of memantine: detection, achieving appropriate column retention, and limited choice of mobile phase components, as a result of memantine molecular structure. Among more than 35 tested columns, the best retention and peak symmetry yielded two C8 and three C18 columns with different characteristics, at a temperature of 30°C, mobile phase composed of 1%, v/v, acetonitrile and 99%, v/v, of 0.05–0.1% phosphoric acid or 2.5–5 mmol phosphate buffer, at flow rate of 1 mL/min and injection volume of 5 µL. The retention time of memantine was between 2.6 and 4 min. Both mobile phase concepts showed perfect linearity, precision, and accuracy. This is the first successful and reproducible direct reverse phase‐high‐performance liquid chromatography–ultraviolet quantification method for memantine.     

A CE‐LIF method based on long wavelength fluorescence labeling for the analysis of thiols in human urine

A rapid and robust CE method using a long wavelength fluorescent reagent 1,7‐dimethyl‐3,5‐distyryl‐8‐phenyl‐(2‐maleimide)difluoroboradiaza‐s‐indacene as the labeling reagent has been developed for the simultaneous determination of thiols, including glutathione, cysteine, homocysteine, N‐acetylcysteine, cysteinylglycine, and penicillamine. The derivatization reaction was carried out in 14 mmol/L pH 8.5 borate buffer at 30°C for 6 min and the labeled thiols derivatives were separated with the running buffer containing 30 mmol/L pH 7.4 phosphate, 30% v/v acetonitrile and 8 mmol/L SDS within 12 min. Detection limits ranged from 0.4 to 2.4 nmol/L. To demonstrate the capability of this method, it was applied to the analysis of thiols in human urine with recoveries of 92.4–105.6%. The derivatization reaction was much faster at milder conditions, and the analysis was rapider. Moreover, with excitation wavelength at long wavelength region, background interference from samples was reduced effectively. The present method seems to be a potential choice for quantifying thiols in human urine.     

The stable o‐phthalaldehyde‐ferrocene/6‐ferrocenyl‐1‐hexanethiol pre‐column derivatization high‐performance liquid chromatography of dimethylarginine by novel dual fluorescence and electrochemical detector

Monomethylarginine, asymmetric dimethylarginine and symmetric dimethylarginine were separated on a Wakopak Combi ODS with an acetonitrile–100 mm potassium phosphate buffer (pH 7.0; 1:1, v/v). Dimethylarginines were derived from o‐phthalaldehyde for the fluorescence detector and from 6‐ferrocenyl‐1‐hexanethiol for the electrochemical detector. The detection limits of the dimethylarginines in spiked plasma were 0.3–0.5 pmol by electrochemical detection and 1–2 pmol by fluorescence detection. The detection limits were improved over 30 times by electrochemical detection and 10 times by fluorescence detection compared with previous reports. In previous derivatization liquid chromatography, the reaction solutions, o‐phthalaldehyde, 2‐mercaptethanol and dimethylarginines were unstable and required quick derivatization at 4°C. By our proposed pre‐column methods, the dimethylarginines were derivatized at room temperature and the fluorescent products were stable for 6 h. The manipulation performance was greatly advanced compared with previous LC reports. This is the first report on stable and sensitive dimethylarginines by dual detection. The selectivity was also improved by dual detection. The proposed method was applied to preliminary monitoring of dimethylargines in plasma and urine. Copyright © 2012 John Wiley & Sons, Ltd.     

Determination of amino acid neurotransmitters in rat hippocampi by HPLC‐UV using NBD‐F as a derivative

A simple, rapid and accurate high‐performance liquid chromatography method with ultraviolet–visible detection was developed for the determination of five amino acid neurotransmitters – aspartate, glutamic acid, glycine, taurine and γ‐aminobutyric acid – in rat hippocampi with pre‐column derivatization with 4‐fluoro‐7‐nitrobenzofurazan. Several conditions which influenced derivatization and separation, such as pH, temperature, acetonitrile percentage mobile phase and flow rate, were optimized to obtain a suitable protocol for amino acids quantification in samples. The separation of the five neurotransmitter derivatives was performed on a C₁₈ column using a mobile phase consisting of phosphate buffer (0.02 mol/L, pH 6.0)–acetonitrile (84:16, v/v) at a flow rate of 1.0 mL/min with the column temperature at 30°C. The detection wavelength was 472 nm. Without gradient elution, the five neurotransmitter derivatives were completely separated within 15 min. The linear relation was good in the range from 0.50 to 500 µmol/L, and the correlation coefficients were ≥0.999. Intra‐day precision was between 1.8 and 3.2%, and inter‐day precision was between 2.4 and 4.7%. The limits of detection (signal‐to‐noise ratio 3) were from 0.02 to 0.15 µmol/L. The established method was used to determine amino acid neurotransmitters in rat hippocampi with satisfactory recoveries varying from 94.9 to 105.2%. Copyright © 2013 John Wiley & Sons, Ltd.     

Rapid and sensitive determination of free thiols by capillary zone electrophoresis with near‐infrared laser‐induced fluorescence detection using a new BODIPY‐based probe as labeling reagent

A CZE with near‐infrared (NIR) LIF detection method has been developed for the analysis of six low molecular weight thiols including glutathione, homocysteine, cysteine, γ‐glutamylcysteine, cysteinylglycine, and N‐acetylcysteine. For this purpose, a new NIR fluorescent probe, 1,7‐dimethyl‐3,5‐distyryl‐8‐phenyl‐(4'‐iodoacetamido)difluoroboradiaza‐s‐indacene was utilized as the labeling reagent, whose excitation wavelength matches the commercially available NIR laser line of 635 nm. The optimum procedure included a derivatization step of the free thiols at 45°C for 25 min and CZE analysis conducted within 14 min in the running buffer containing 16 mmol/L pH 7.0 sodium citrate and 60% v/v ACN. The LODs (S/N = 3) ranged from 0.11 nmol/L for N‐acetylcysteine to 0.31 nmol/L for γ‐glutamylcysteine, which are better than or comparable to those reported with other derivatization‐based CE‐LIF methods. As the first trial of NIR CE‐LIF method for thiol determination, the practical application of the proposed method has been validated by detecting thiols in cucumber and tomato samples with recoveries of 96.5–104.3%.     

Quantification of L‐ergothioneine in whole blood by hydrophilic interaction ultra‐performance liquid chromatography and UV‐detection

A new hydrophilic interaction ultra‐performance LC method was established for the whole blood measurement of L‐ergothioneine. Chromatographic separation was achieved in a fairly short time, less than 4 min, on a 100 × 2.1 mm Acquity UPLC BEH HILIC 1.7 μm column with a mobile phase consisting of a mixture of 100 mmol/L ammonium acetate/ACN/water (5:85:10, v/v/v) that flowed isocratically at 0.250 mL/min. The LOD and the limit of quantification were 3.85 and 11.67 μmol/L, respectively. The method exhibited linearity in a concentration range of 15.63–1000 μmol/L (R² > 0.999). Mean recovery was 96.34% whereas intraassay and interassay precision were 1.52 and 1.82% RSD, respectively. On the whole, the developed method is simple, fast, precise, accurate, and sensitive and may be useful for routine analyses.     

Separation of structurally related primary aliphatic amines using hydrophilic interaction chromatography with fluorescence detection after postcolumn derivatization with o‐phthaldialdehyde/mercaptoethanol

The retention behavior of primary aliphatic amines (homologous series of aliphatic alkyl amines and cycloalkyl amines) and positional isomers of alkylamines in the hydrophilic interaction chromatography mode was studied. The study was carried out on a TSKgel Amide‐80 column followed by postcolumn derivatization with fluorescence detection to describe the retention mechanism of tested compounds. The effect of chromatographic conditions including column temperature, acetonitrile content in the mobile phase, mobile phase pH (ranging from 3.5 to 6.8), and salt concentration in the mobile phase was investigated. The final mobile phase consisted of acetonitrile and solution of 20 mM potassium formate pH 3.5 in ratio 80:20 v/v. The analyses were carried out at mobile phase flow rate of 1.0 mL/min and the column temperature of 20°C. The developed method was fully validated in terms of linearity, sensitivity (limit of detection and limit of quantification), accuracy, and precision according to International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. The proposed new methods were proved to be highly sensitive, simple, and rapid, and were successfully applied to the determinations of isopropylamine, cyclohexylamine, and cyclopropylamine in relevant active pharmaceutical ingredients.     

3‐(2‐Bromoacetamido)‐N‐(9‐ethyl‐9H)‐carbazol fluorescent probe and its application for the determination of thiophenols in rubber products by HPLC with fluorescence detection and atmospheric chemical ionization mass spectrometry identification

A rapid, sensitive, and selective precolumn derivatization method for the simultaneous determination of eight thiophenols using 3‐(2‐bromoacetamido)‐N‐(9‐ethyl‐9H )‐carbazol as a labeling reagent by high‐performance liquid chromatography with fluorescence detection has been developed. The labeling reagent reacted with thiophenols at 50°C for 50 min in aqueous acetonitrile in the presence of borate buffer (0.10 mol/L, pH 11.2) to give high yields of thiophenol derivatives. The derivatives were identified by online postcolumn mass spectrometry. The collision‐induced dissociation spectra for thiophenol derivatives gave the corresponding specific fragment ions at m/z 251.3, 223.3, 210.9, 195.8, and 181.9. At the same time, derivatives exhibited intense fluorescence with an excitation maximum at λ_ex = 276 nm and an emission maximum at λ_em = 385 nm. Excellent linear responses were observed for all analytes over the range of 0.033–6.66 μmol/L with correlation coefficients of more than 0.9997. Detection limits were in the range of 0.94–5.77 μg/L with relative standard deviations of less than 4.54%. The feasibility of derivatization allowed the development of a rapid and highly sensitive method for the quantitative analysis of trace levels of thiophenols from some rubber products. The average recoveries (n = 3) were in the range of 87.21–101.12%.     

HILIC LC‐MS for the determination of 2′‐C‐methyl‐cytidine‐triphosphate in rat liver

A very accurate and selective LC‐MS/MS method was developed and validated for the quantification of 2′‐C‐modified nucleoside triphosphate in liver tissue samples. An efficient pretreatment procedure of liver tissue samples was developed, using a fully automated SPE procedure with 96‐well SPE plate (weak anion exchange sorbent, 30 mg). Nucleotide hydrophilic interaction chromatography has been performed on an aminopropyl column (100 mm×2.0 mm, 3 μm) using a gradient mixture of ACN and ACN/water (5:95 v/v) with 20 mM ammonium acetate at pH 9.45 as mobile phase at 300 μL/min flow rate. The 2′‐C‐modified nucleoside triphosphate was detected in the negative ESI mode in multiple reaction monitoring (MRM) mode. Calibration curve was linear over the 0.05–50 μM concentration range. Satisfying results, confirming the high reliability of the established LC‐MS/MS method, were obtained for intraday precision (CV = 2.5–9.1%) and accuracy (92.6–94.8%) and interday precision (CV = 9.6–11.5%) and accuracy (94.4–102.4%) as well as for recovery (82.0–112.6%) and selectivity. The method has been successfully applied for pharmacokinetic studies of 2′‐C‐methyl‐cytidine‐triphosphate in liver tissue samples.     

Quantitative determination of seven chemical constituents and chemo‐type differentiation of chamomiles using high‐performance thin‐layer chromatography

A simple and rapid high‐performance thin‐layer chromatographic method was developed for the separation and determination of six flavonoids (rutin, luteolin‐7‐O‐β‐glucoside, chamaemeloside, apigenin‐7‐O‐β‐glucoside, luteolin, apigenin) and one coumarin, umbelliferone from chamomile plant samples and dietary supplements. The separation was achieved on amino silica stationary phase using dichloromethane/acetonitrile/ethyl formate/glacial acetic acid/formic acid (11:2.5:3:1.25:1.25 v/v/v/v/v) as the mobile phase. The quantitation of each compound was carried out using densitometric reflection/absorption mode at their respective absorbance maxima after postchromatographic derivatization using natural products reagent (1% w/v methanolic solution of diphenylboric acid‐β‐ethylamino ester). The method was validated for specificity, limits of detection and quantification, precision (intra‐ and interday) and accuracy. The limits of detection and quantification were found to be in the range from 6–18 and 16–55 ng/band for six flavonoids and one coumarin, respectively. The intra‐ and interday precision was found to be <5% RSD and recovery of all the compounds was >90%. The data acquired from high‐performance thin‐layer chromatography was processed by principal component analysis using XLSTAT statistical software. Application of principal component analysis and agglomerative hierarchial clustering was successfully able to differentiate two chamomiles (German and Roman) and Chrysanthemum.     

Determination of paroxetine in serum treated with simple pretreatment by pre‐column high‐performance liquid chromatography using 4‐(5,6‐dimethoxy‐2‐phthalimidinyl)‐2‐methoxyphenylsulfonyl chloride as a fluorescent labeling reagent

The therapeutic drug monitoring of paroxetine could be used to optimize the pharmacological treatment of depressed patients. A simple and sensitive high‐performance liquid chromatography procedure was developed for the determination of paroxetine in serum. After simple pretreatment of serum (50 μL) with acetonitrile and o‐phthalaldehyde, paroxetine was derivatized with 4‐(5,6‐dimethoxy‐2‐phthalimidinyl)‐2‐methoxyphenylsulfonyl chloride at 70°C for 20 min in borate buffer (0.1 mol/L, pH 8.0) to produce a fluorescent product. The derivative was separated on a reversed‐phase column at 40°C for stepwise elution with (A) acetic acid (10 mmol/L) and (B) acetonitrile. The flow rate was 1.0 mL/min. The fluorescence intensity was monitored at excitation and emission wavelengths of 320 and 400 nm, respectively. The within‐day and day‐to‐day relative standard deviations were 3.0–3.4 and 2.7–8.3%, respectively. The detection limit of paroxetine was 8.3 fmol at a signal‐to‐noise ratio of 3. As the proposed method that only requires a small quantity of serum (50 μL) is simple, sensitive and reproducible, it would be useful for clinical and biochemical research as well as drug monitoring. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous determination of three anticon‐vulsants using hydrophilic interaction LC‐MS

A specific and automated method was developed to quantify the anticonvulsants gabapentin, pregabalin and vigabatrin simultaneously in human serum. Samples were prepared with a protein precipitation. The hydrophilic interaction chromatography (HILIC) with a mobile phase gradient was used to divide off ions of the matrix and for separation of the analytes. Four different HILIC‐columns and two different column temperatures were tested. The Tosoh‐Amid column gave the best results: single small peaks. The anticonvulsants were detected in the multiple reaction monitoring mode (MRM) with ESI‐MS‐MS. Using a volume of 100 μL biological sample the lowest point of the standard curve, i.e. the lower LOQs were 312 ng/mL. The described HILIC‐MS‐MS method is suitable for therapeutic drug monitoring and for clinical and pharmcokinetical investigations of the anticonvulsives.     

Application of pH‐zone‐refining countercurrent chromatography in the chiral separation of two β‐adrenergic blocking agents

Two β‐adrenergic blocking agents, 1‐[(1‐methylethyl)amino]‐3‐phenoxy‐2‐propanol ( 1 ) and 1‐[(1‐methylethyl)amino]‐3‐(3‐methylphenoxy)‐2‐propanol ( 2 ; Toliprolol), were enantioseparated by pH‐zone‐refining countercurrent chromatography. A two‐phase solvent system composed of chloroform containing 0.10 mol/L of di‐n‐hexyl l‐ tartrate/0.10 mol/L of boric acid aqueous solution (1:1, v/v) was selected, in which 20 mmol/L triethylamine was added in the organic phase as a retainer and 2 mmol/L HCl was added in the aqueous phase as an eluter. Fifty milligrams of each racemate was completely enantioseparated by pH‐zone‐refining countercurrent chromatography to yield each enantiomer with a purity of more than 98%, and the recovery of each separated enantiomer reached around 76–82%.     

Reversed‐phase high‐performance liquid chromatography method with fluorescence detection to screen nitric oxide synthases inhibitors

Nitric oxide synthase (NOS) inhibitors are potential drug candidates due to the critical role of an excessive production of nitric oxide in a range of diseases. At present, the radiometric detection of l ‐[³H]‐citrulline produced from l ‐[³H]‐arginine during the enzymatic reaction is one of the most accepted methods to assess the in vitro activity of NOS inhibitors. Here we report a fast, easy, and cheap reversed‐phase high‐performance liquid chromatography method with fluorescence detection, based on the precolumn derivatization of l ‐citrulline with o‐phthaldialdehyde/N‐acetyl cysteine, for the in vitro screening of NOS inhibitors. To evaluate enzyme inhibition by the developed method, N‐[3‐(aminomethyl)benzyl]acetamidine, a potent and selective inhibitor of inducible NOS, was used as a test compound. The half maximal inhibitory concentration obtained was comparable to that derived by the well‐established radiometric assay.     

pH‐zone‐refining elution–extrusion countercurrent chromatography: Separation of hydroxyanthraquinones from Cassiae semen

Seven hydroxyanthraquinones were successfully separated from the traditional Chinese medicinal herb Cassiae semen by conventional and pH‐zone‐refining countercurrent chromatography with an environmentally friendly biphasic solvent system, in which elution–extrusion mode was investigated for pH‐zone‐refining countercurrent chromatography for the first time. A two‐phase solvent system composed of n‐hexane/ethyl acetate/ethanol/water (5:3:4:4, v/v/v/v) was used for the conventional countercurrent chromatography while the same system with a different volume ratio n‐hexane/ethyl acetate/ethanol/water (3:5:2:6, v/v/v/v) was used for pH‐zone‐refining countercurrent chromatography, in which 20 mmol/L of trifluoroacetic acid was added in the organic phase as a retainer and 15 mmol/L of ammonia was added to the aqueous phase as an eluter. A 400 mg crude sample could be well separated by pH‐zone‐refining countercurrent chromatography, yielding 53 mg of aurantio‐obtusin, 40 mg of chryso‐obtusin, 18 mg of obtusin, 24 mg of obtusifolin, 10 mg of emodin, and 105 mg of the mixture of chrysophanol and physcion with a purity of over 95.8, 95.7, 96.9, 93.5, 97.4, 77.1, and 19.8%, as determined by high‐performance liquid chromatography. Furthermore, the difference in elution sequence between conventional and pH‐zone‐refining mode was observed and discussed.     

Quantitative analysis of ripasudil hydrochloride hydrate and its impurities by reversed‐phase high‐performance liquid chromatography after precolumn derivatization: Identification of four impurities

We report the development and validation of a stability‐indicating reversed‐phase high‐performance liquid chromatography method with precolumn derivatization for the separation and identification of the impurities of ripasudil hydrochloride hydrate, a novel protein kinase inhibitor. 2,3,4,6‐Tetra‐O‐acetyl‐β‐d ‐glucopyranosyl isothiocyanate was chosen as the derivatizing reagent and triethylamine was added as catalyst. 200 μL sample solution (1 mg/mL), 600 μL derivatizing reagent (1 mg/mL), and 200 μL triethylamine solution (1%, v/v) were mixed and reacted at 40°C for 30 min. The separation was achieved on an Inertsil C₁₈ ODS‐3 (250 mm × 4.6 mm, 5 μm) column using mobile phases including 10 mmol monopotassium phosphate buffer (pH 3.0) and methanol in gradient mode. The column temperature was adjusted at 25°C and the flow rate at 1 mL/min. The detection was carried out at 220 nm. Different precolumn derivatization conditions as well as the high‐performance liquid chromatography conditions were optimized. Ripasudil hydrochloride hydrate and its four impurities were detected and quantitated, among which two new compounds were characterized. The proposed method was validated and proven to be selective, accurate, and precise and suitable for the quantitative analysis of ripasudil hydrochloride hydrate.     

Determination of glutathione and cysteine in yeasts by hydrophilic interaction liquid chromatography followed by on‐line postcolumn derivatization

In the present study, we report a new method for the determination of two primary thiols, cysteine (CYS) and glutathione (GSH), by hydrophilic interaction LC. The polar analytes are separated isocratically using a mobile phase consisting of 65% acetonitrile/35% ammonium acetate (15 mmol/L, pH 2.0) and are detected at 285 nm following on‐line postcolumn derivatization by the thiol‐selective reagent methyl propiolate. The main figures of merit included linearity in the range of 5–200 μmol/L and an LOD 0.6 μmol/L for both compounds. The absence of matrix effect allowed the determination of CYS and GSH in various yeast samples. GSH was present in most of the samples at levels ranging between 0.9 and 3.1 mg/g, whereas CYS was determined in only one sample at a significantly lower concentration. In terms of accuracy, the percent recoveries ranged between 91.2 and 105.6% for GSH, and 91.6 and 106.9% for CYS.     

Analysis of Morpholinium Ionic Liquid Cations by Hydrophilic Interaction Columns Coupled with Indirect UV Detection

A method was developed for the separation and detection of morpholinium ionic liquid cations by hydrophilic interaction column combined with indirect ultraviolet detection using imidazolium ionic liquids as ultraviolet absorbents in high‐performance liquid chromatography. The effects of the ultraviolet absorbents, organic solvents, and the pH value of the aqueous solution in the mobile phase for the determination of morpholinium cations were investigated by using a hydrophilic column with carbamoyl group as the analytical column. The retention and detection behavior of morpholinium cations was discussed. The suitable chromatographic conditions were 0.8 mmol/L 1‐ethyl‐3‐methyl‐imidazolium tetrafluoroborate aqueous solution (pH 3.5 adjusted with acetic acid)/acetonitrile (45/55, v/v) as the mobile phase and a detection wavelength of 210 nm. Under these conditions, the baseline separation of N‐methyl‐N‐ethyl‐morpholinium cation ([MEMo]⁺) and N‐methyl‐N‐propyl‐morpholinium cation ([MPMo]⁺) was successfully achieved in 15 min. The detection limits of [MEMo]⁺ and [MPMo]⁺ were 0.595 and 0.531 mg/L, respectively. Relative standard deviations were less than 0.2%. This method has been successfully applied to the analysis of morpholinium ionic liquid samples synthesized in chemical laboratories, which is simple, reliable, and practical.