LC-MS Determination and Pharmacokinetic Study of Luteolin-7-O-β-d-glucoside in Rat Plasma after Administration of the Traditional Chinese Medicinal Preparation Kudiezi Injection

A rapid and sensitive LC-MS method has been developed for the determination of luteolin-7-O-β-d-glucoside in rat plasma after solvent extraction. Separation was on an Elite Hypersil ODS2 column (250 mm × 4.6 mm i.d., 5 μm) with a mobile phase of acetonitrile-0.3% acetic acid (26:74, v/v). The samples were analyzed by using positive electrospray ionization MS in selected ion monitoring mode. The selected ions for luteolin-7-O-β-d-glucoside and the internal standard, isoquercitrin, were m/z 448.95 and m/z 464.95. Good linearity was observed over the range of 20–2,000 ng mL^?1 with a lower limit of quantification of 20 ng mL^?1. No interference peaks or matrix effects were observed. The validated method was applied to the pharmacokinetic study of luteolin-7-O-β-d-glucoside in rat plasma after intravenous administration of Kudiezi Injection.     

Comparison of HPLC Versus HPTLC-Densitometry for the Quantification of Chromone Glucosides in Saposhnikoviae divaricatae Radix

A HPLC and a HPTLC-densitometric method were developed for the quantification of prim-O-glucosylcimifugin and 4′-O-β-d-glucosyl-5-O-methylvisamminol the major chromone glucosides in the roots of Saposhnikovia divaricata. The validation of both methods resulted in comparable parameters regarding stability, specificity, linearity, robustness, precision and recovery, whereas complementary advantages were obtained concerning LOD and LOQ. The HPTLC-based densitometry revealed a lower LOD (1.11 versus 4.37 μg mL⁻¹ in HPLC) and LOQ (3.36 versus 13.24 μg mL⁻¹ in HPLC) for prim-O-glucosylcimifugin, whereas the HPLC resulted in a lower LOD (1.00 versus 4.10 μg mL⁻¹ in HPTLC-densitometry) and LOQ (3.04 versus 12.46 μg mL⁻¹ in HPTLC-densitometry) for 4′-O-β-d-glucosyl-5-O-methylvisamminol. Both methods revealed nearly matching contents of the chromones after analysis of different commercially available batches of Saposhnikoviae divaricatae radix with a total content for both chromone glycosides in the range from 0.31 ± 0.011 to 0.56 ± 0.021 % determined by HPLC and between 0.34 ± 0.011 and 0.61 ± 0.009 % determined by HPTLC. The plant material cultivated in Germany showed a very similar content and ratio of both chromone glucosides in comparison to the standard batches originating from China.

     

Simultaneous Determination of Safingol and d-erythro-Sphinganine in Human Plasma by LC with Fluorescence Detection

l-threo-Sphinganine (safingol) is a putative synthetic sphingosine kinase inhibitor currently being tested in clinical trials as an anticancer agent. To enable defining the pharmacokinetic properties of safingol in humans, we developed a sensitive analytical method to simultaneously quantitate safingol and its naturally-occurring diastereomer, d-erythro-sphinganine in human plasma. Of the two different fluorogenic derivatization agents (NDA and OPA) and several pH conditions compared for the derivatization, we found that NDA derivatization achieved more than 20 times greater sensitivity compared with OPA derivatization, and pH 9.0 showed the highest sensitivity for both compounds. An analytical method for liquid chromatography (LC) with a fluorescence detector (FLD) was developed and validated with calibration curve ranges of 20–1,000 ng mL⁻¹ for safingol and d-erythro-sphinganine. Our LC-FLD method using NDA-derivatization enabled simultaneous quantification of safingol and its naturally-occurring diastereomer, d-erythro-sphinganine with satisfactory sensitivity in human plasma.

     

Quantitative Analysis of Caffeoylquinic Acids and Styrylpyrones in Sweetia panamensis Bark by UPLC

Six secondary metabolites from the methanolic extract of Sweetia panamensis (Fabaceae) bark were isolated and characterised. Along with the pyrones desmethylangonine β-d-O-glucopyranoside and desmethylangonine β-d-O-glucopyranosyl-(1→6)-O-β-d-glucopyranoside, already reported in this species, 5-O-caffeoylquinic acid (chlorogenic acid), 4-O-caffeoylquinic acid, 3-O-caffeoylquinic acid and the isoflavonoid 5-O-methylgenistein 7-O-β-d-glucopyranoside were isolated for the first time from S. panamensis. Additionally, an LC-ESI-MS qualitative analysis was performed and an ultra performance liquid chromatography (UPLC) method was developed and validated for the determination of these compounds. The UPLC method was applied to the quantitative analysis of plant samples. Pyrones and caffeoylquinic acids resulted to be the main compounds in the extract; in particular desmethylangonine β-d-O-glucopyranosyl-(1→6)-O-β-d-glucopyranoside was the most abundant compound.

     

UPLC-PDA Analysis for Simultaneous Quantification of Four Active Compounds in Crude and Processed Rhizome of Polygonum multiflorum Thunb

A novel, rapid and specific ultra performance liquid chromatography-photo diode array detection method was developed for the simultaneous determination of 2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside (TSG), emodin-8-O-β-d-glucoside (EMG), emodin (EM) and physcion (PS). The chromatographic separation was performed on an Acquity BEH C₁₈ column (100 × 2.1 mm i.d., 1.7 μm). The mobile phase was a mixture of 0.3% acetic acid–water and 0.3% acetic acid–acetonitrile employing gradient elution at the flow rate of 0.4 mL min^?1. The four compounds behaved linearly in the concentration range between 60.80–3040.00 μg mL^?1 (TSG), 0.50–25.00 μg mL^?1 (EMG), 2.16–108.00 μg mL^?1 (EM) and 1.56–78.00 μg mL^?1 (PS), respectively with correlation coefficients >0.999. The precision of the method were below 5% RSD. Recoveries of the four compounds ranged from 95.71 to 102.97%, with RSD values less than 2%.     

Preparative Isolation and Purification of Flavonoids and Protocatechuic Acid from Sea Buckthorn Juice Concentrate (Hippophaë rhamnoides L. ssp. rhamnoides) by High-Speed Counter-Current Chromatography

High-speed counter-current chromatography (HSCCC)—a support free all liquid–liquid chromatography technique—has been successfully used for the preparative isolation of isorhamnetin 3-O-β-d-glucoside, isorhamnetin 3-O-β-rutinoside, quercetin 3-O-β-d-glucoside, syringetin 3-O-β-d-glucoside and protocatechuic acid from sea buckthorn juice concentrate (Hippophaë rhamnoides L. ssp. rhamnoides, Elaeagnaceae). The preparative HSCCC instrument was a multilayer coil planet centrifuge equipped with three preparative coils. Separation was performed with a two phase solvent system (n-hexane–n-butanol–water, 1:1:2 v/v/v) in ‘head-to-tail’ mode. Each injection of 4.1 g crude ethyl acetate extract yielded isorhamnetin 3-O-β-d-glucoside (95 mg), isorhamnetin 3-O-β-rutinoside (10 mg), quercetin 3-O-β-d-glucoside (5 mg), and protocatechuic acid (34 mg) with purities >98%. The flavonoid syringetin 3-O-β-d-glucoside (2 mg) was a novel compound for H. rhamnoides. Chemical structures of all compounds were determined by HPLC–ESI–MS–MS, 1D-NMR (¹H, ¹³C, DEPT 135) spectroscopy and for elucidation of glycosidic linkages 2D-NMR (HMBC) spectroscopy was used.     

Anti-influenza effect of the major flavonoids from Salvia plebeia R.Br. via inhibition of influenza H1N1 virus neuraminidase

To determine the compounds responsible for its anti-influenza activities, we isolated the three flavonoids, 6-hydroxyluteolin 7-O-β-d-glucoside (1), nepitrin (2), homoplantaginin (3) from the MeOH extract of Salvia plebeia R.Br. and identified them by comparing the spectroscopic data with that reported in the literature. The contents of the three flavonoids in the whole extract were 108.74 ± 0.95, 46.26 ± 2.19, and 69.35 ± 1.22 mg/g for 6-hydroxyluteolin 7-O-β-d-glucoside, nepitrin, and homoplantaginin, respectively, which demonstrates that they are the major constituents of this plant. The three flavonoids were evaluated for their inhibitory activities against influenza virus H1N1 A/PR/9/34 neuraminidase and H1N1-induced cytopathic effect (CPE) on Madin-Darby canine kidney (MDCK) cells. Our results demonstrated the following arrangement for their anti-influenza activities: nepitrin (2) > 6-hydroxyluteolin 7-O-β-d-glucoside (1) > homoplantaginin (3). The potent inhibitory activities of these flavonoids against influenza suggested their potential to be developed as novel anti-influenza drugs in the future.     

The Development and Validation of a Stability-Indicating UHPLC-DAD Method for Determination of Perindopril l-Arginine in Bulk Substance and Pharmaceutical Dosage Form

A stability-indicating ultra-high-performance liquid chromatography (UHPLC) method with a diode array detector was developed and validated for the determination of cis/trans isomers of perindopril l-arginine in bulk substance and pharmaceutical dosage form. The separation was achieved on a Poroshell 120 Hilic (4.6 × 150 mm, 2.7 µm) column using a mobile phase composed of acetonitrile–0.1 % formic acid (20:80 v/v) at a flow rate of 1 mL min⁻¹. The injection volume was 5.0 µL and the wavelength of detection was controlled at 230 nm. The selectivity of the UHPLC-DAD method was confirmed by determining perindopril l-arginine in the presence of degradation products formed during acid–base hydrolysis and oxidation as well as degradation in the solid state, at an increased relative air humidity and in dry air. The method’s linearity was investigated in the ranges 0.40–1.40 µg mL⁻¹ for isomer I and 0.40–2.40 µg mL⁻¹ for isomer II of perindopril l-arginine. The UHPLC-DAD method met the precision and accuracy criteria for the determination of the isomers of perindopril l-arginine. The limits of detection and quantitation were 0.1503 and 0.4555 µg mL⁻¹ for isomer I and 0.0356 and 0.1078 µg mL⁻¹ for isomer II, respectively.

     

A new acylated flavonoid tetraglycoside with anti-inflammatory activity from Tipuana tipu leaves

A new acylated kaempferol glycoside, kaempferol 3-O-α-l-rhamnopyranosyl-(1 → 6)-O-[β-d-glucopyranosyl-(1 → 2)-4-O-acetyl-α-l-rhamnopyranosyl-(1 → 2)]-β-d-galactopyranoside, has been isolated from the leaves of Tipuana tipu (Benth.) Lillo growing in Egypt, along with three known flavonol glycosides, kaempferol 3-O-rutinoside, quercetin 3-O-rutinoside (rutin) and kaempferol 3-O-[α-l-rhamnopyranosyl-(1 → 6)]-[α-l-rhamnopyranosyl-(1 → 2]-β-d-glucopyranoside. Structure elucidation was achieved through different spectroscopic methods. Structure relationship with anti-inflammatory activity using carrageenin-induced rat paw oedema model is discussed.     

Separation and Characterization of Unknown Impurities in Amikacin Sulfate by HPLC–MS n

Nine impurities in amikacin sulfate made in China were separated and identified by HPLC–MSⁿ for the further improvement of official monographs in pharmacopoeias. The mass fragmentation patterns and structural assignment of these impurities were studied. The column was Acchrom Click XIon (250 × 4.6 mm, 5 μm). The mobile phase was 250 m mol L⁻¹ ammonium formate and 1.4 % formic acid aqueous solution–acetonitrile–water (30:48:22). In positive mode, full scan LC–MS was first performed in order to obtain the m/z value of the protonated molecules, LC–MS–MS was then carried out on the compounds of interest on AB SCIEX 4000 Q TRAP™ composite triple quadrupole/linear ion trap tandem mass spectrometer. The complete fragmentation patterns of nine impurities were studied and used to obtain information about the structure of these impurities. The structures of nine impurities in amikacin sulfate were deduced based on the HPLC–MSⁿ data, in which three impurities were novel impurities. Three novel impurities were 1-N-(l-4-amino-2-hydroxybutyryl) derivative of 4-O-(6-AG)DS, 1-N-(l-4-amino-2-hydroxybutyryl) derivative of 6-O-(3-AG)DS and 1-N-(l-4-amino-2-hydroxybutyryl) derivative of kanamycin D.

     

New flavonol glycosides from the leaves of Caragana brachyantha

Two new flavonol glycosides, brachysides C and D, together with three known flavonol glycosides, were isolated from the leaves of Caragana brachyantha. The structures of brachysides C and D were elucidated on the basis of detailed spectroscopic analysis as quercetin 5-O-[α-l-rhamnopyranosyl-(1 → 6)-β-d-glucopyranoside]-7-O-[α-l-rhamnopyranoside] and quercetin 5-O-[α-l-rhamnopyranosyl-(1 → 6)-β-d-glucopyranoside]-7-O-[α-l-rhamnopyranoside]-4′-O-[α-l-rhamnopyranoside], respectively. The presence of flavonol tetra- and triglycosides bearing a sugar moiety at position 5 was the first report from this genus Caragana.     

Fast and Precise Quantification of l-Homoarginine in Human Plasma by HILIC-Isotope Dilution-MS–MS

Lowered plasma concentrations of the endogenous amino acid l-homoarginine have been recently identified as an independent risk factor for cardiovascular and all-cause mortality in patients referred for coronary angiography in the LURIC study. To support further investigations into this matter, we describe here a fast and easy LC–MS–MS method for the detection of l-homoarginine in human plasma. The sample preparation consisted only of the addition of the stable isotope-labeled internal standard d ₄-l-homoarginine and protein precipitation. The analytes were separated isocratically on an HILIC silica column. Detection took place by tandem mass spectrometry. The calibration function was linear in the range of 0.1–10 μmol L⁻¹. The intra-day precision was better than 2 % RSD and the inter-day precision better than 4 % RSD in plasma. The accuracy was always better than 5 % deviation. The method was matrix independent owing to the usage of the analogous stable isotope-labeled internal standard.

     

Two new quercetin glycoside derivatives from the fruits of Gardenia jasminoides var. radicans

Two new quercetin glycoside derivatives named quercetin-3-O-[2-O-trans-caffeoyl-α-l-rhamnopyranosyl-(1 → 6)-β-d-glucopyranoside] (1) and quercetin-3-O-[2-O-trans-caffeoyl-β-l-rhamnopyranosyl-(1 → 6)-β-d-glucopyranoside] (2) along with three known flavonoids, 5-hydroxy-6,7,3′,4′,5′-pentamethoxyflavone (3), 5,7-dihydroxy-8-methoxyflavone (4) and kaempferol 3-O-β-d-glucopyranoside (5), were isolated from the fruits of Gardenia jasminoides var. radicans. The structures of the new compounds were determined by means of extensive spectroscopic analysis (1D, 2D NMR and HR-ESI-MS), glycoside hydrolysis and sugar HPLC analysis after derivatisation. This is the first report on the isolation of a pair of compounds with α or β-l-rhamnopyranosyl configuration from plant and the first detail assignment of their NMR data.     

Enantioselective Separation and Determination of Carnosine in Rat Plasma by Fluorescence LC for Stereoselective Pharmacokinetic Studies

A sensitive fluorescence liquid chromatographic analytical method was developed for the simultaneous determination of carnosine enantiomers in rat plasma. The method was applied to pharmacokinetic studies. Chiral separation of carnosine enantiomers was achieved by pre-column derivatization with o-phthaldialdehyde and the thiol N-acety-l-cysteine as derivating reagents. They were separated on an ODS column and detected by fluorescence detection (λ_ex = 350 nm, λ_em = 450 nm). γ-Aminobutyric acid was used as internal standard. The method was linear up to 6,000 ng mL^?1 for l-carnosine, 4,000 ng mL^?1 for d-carnosine. Low limit of quantitation (LLOQ) was 40 ng mL^?1 for each isomer. The relative standard deviations obtained for intra- and inter-day precision were lower than 12% and the recoveries were higher than 75% for both enantiomers. The method was applied to a stereoselective study on the pharmacokinetics of carnosine after oral administration with a single dose (carnosine, 75 mg kg^?1 for each isomer) to a rat. The initial data indicated that l-carnosine had a larger value of the highest plasma concentration than d-carnosine (C _max 5,344 vs. 1,914 ng mL^?1), and that of l-carnosine had a lower value of AUC_(0?∞) and t _1/2(h) (AUC_(0?∞) 5,306 vs. 6,321 ng h mL^?1, t _1/2 1.43 vs. 3.37 h). Our results indicated that the pharmacokinetic of l-carnosine and d-carnosine revealed enantioselective properties significantly.     

Simultaneous Analysis of Zofenopril and Its Active Metabolite Zofenoprilat in Human Plasma by LC–ESI-MS Using Pre-Column Derivatization with p-Bromophenacyl Bromide

Zofenoprilat is an active metabolite of zofenopril, which is very unstable in plasma because of oxidative degradation of its thiol group. In this method, p-bromophenacyl bromide was used as derivatization reagent, immediately after plasma separation, to react with the free thiol group of zofenoprilat and form the derivative zofenoprilat-p-BPB. After acidification with 50% acetic acid, the derivatized plasma samples were extracted with methyl tert-butyl ether and separated on a C₁₈ column with 40:60 (v/v) 10 mM ammonium acetate buffer solution containing 0.1% formic acid–acetonitrile as mobile phase. Calibration plots were linear over the concentration range 1–500 ng mL⁻¹ for zofenopril and 2–1,800 ng mL⁻¹ for zofenoprilat. The method was successfully used to study the bioavailability of zofenopril calcium capsules relative to that of zofenopril calcium tablets in healthy Chinese volunteers.

     

Comparison of HPLC Versus HPTLC-Densitometry for the Quantification of Chromone Glucosides in Saposhnikoviae divaricatae Radix

A HPLC and a HPTLC-densitometric method were developed for the quantification of prim-O-glucosylcimifugin and 4′-O-β-d-glucosyl-5-O-methylvisamminol the major chromone glucosides in the roots of Saposhnikovia divaricata. The validation of both methods resulted in comparable parameters regarding stability, specificity, linearity, robustness, precision and recovery, whereas complementary advantages were obtained concerning LOD and LOQ. The HPTLC-based densitometry revealed a lower LOD (1.11 versus 4.37 μg mL^?1 in HPLC) and LOQ (3.36 versus 13.24 μg mL^?1 in HPLC) for prim-O-glucosylcimifugin, whereas the HPLC resulted in a lower LOD (1.00 versus 4.10 μg mL^?1 in HPTLC-densitometry) and LOQ (3.04 versus 12.46 μg mL^?1 in HPTLC-densitometry) for 4′-O-β-d-glucosyl-5-O-methylvisamminol. Both methods revealed nearly matching contents of the chromones after analysis of different commercially available batches of Saposhnikoviae divaricatae radix with a total content for both chromone glycosides in the range from 0.31 ± 0.011 to 0.56 ± 0.021 % determined by HPLC and between 0.34 ± 0.011 and 0.61 ± 0.009 % determined by HPTLC. The plant material cultivated in Germany showed a very similar content and ratio of both chromone glucosides in comparison to the standard batches originating from China.     

Enantioseparation of Three β-Agonists Using Di-n-butyl d-Tartrate-Boric Acid Complex as Chiral Selector by Means of MEEKC

In this study, a self-prepared complex chiral selector, di-n-butyl d-tartrate-boric acid complex, by the reaction of di-n-butyl d-tartrate with boric acid in a running buffer was used as a chiral selector for the enantioseparation of three β-agonists including clenbuterol, cycloclenbuterol and tulobuterol by means of microemulsion electrokinetic chromatography (MEEKC). Three β-agonists were successfully enantioseparated using the chiral system, indicating that the di-n-butyl d-tartrate-boric acid complex was a useful chiral selector. The effects of di-n-butyl d-tartrate and sodium tetraborate concentration, surfactant concentration, cosurfactant, phosphate, buffer pH and composition, as well as applied voltage were extensively investigated to achieve a good enantioseparation. The di-n-butyl d-tartrate and sodium tetraborate concentration in the running buffer had great influence on the chiral resolution (R _s). Three β-agonists which could not be separated with only di-n-butyl d-tartrate, obtained good chiral separation using the complex chiral selector; among them, two pairs including clenbuterol and cycloclenbuterol could be baseline resolved in 7 min under optimized experimental conditions of 0.8% (w/v) di-n-butyl d-tartrate, 40 mM sodium tetraborate, 3.0% (w/v) Tween-20 and 60 mM sodium dihydrogen phosphate with 25 kV as running voltage. The results indicated that the method could be used for the enantioseparation of three β-agonists.

     

Determination of Tryptophan in Lysozyme and Lysozyme Hydrolysate by Chiral LC Using d-Tryptophan as Internal Standard

In the present study, a new LC method is described for the quantitation of tryptophan (Trp) in lysozyme and enzymatic lysozyme hydrolysate. To compensate for partial breakdown of Trp during hydrolysis with 4 M methanesulfonic acid, an enantiomer dilution method was developed. The method makes use of free d-Trp or a d-Trp-containing dipeptide as internal standard for the quantitation of l-tryptophan in these matrices. After acid hydrolysis in 4 M methanesulfonic acid, LC analysis is performed on a Crownpak CR chiral column in combination with fluorescence detection. Optimum time and temperature for the acid hydrolysis were investigated in order to obtain complete hydrolysis of the source materials. A comparison of the l-Trp recoveries was made for d-Trp and Gly-d-Trp as internal standards. By choosing a hydrolysis time of 150 min at 150 °C, 93% recovery of l-Trp from lysozyme was achieved. Under these conditions, no racemization occurred. When choosing d-Trp as internal standard, a direct LC method for l-Trp in lysozyme and enzymatic lysozyme hydrolysate was established without the need for pre-column derivatization and without the need to use Trp protecting agents during acid hydrolysis.