Quantitative analysis of cytokinins in plants by high performance liquid chromatography: electronspray ionization ion trap mass spectrometry

The present paper introduces a highly sensitive and selective method for simultaneous quantification of 12 cytokinins(free form and their conjugates).The method includes a protocol of extraction with methanol/water/formic acid(1514/1,v/v/v)to the micro-scale samples,pre-purification with solid phase extraction(SPE)cartridges of the extracts,separation with a high performance liquid chromatography(HPLC)and detection by an electrospray ionization ion trap mass spectrometry(ESI-Ion trap-MS)system in a consecutive ion monitoring(CRM)mode at the three stage fragmentation of mass spectrometry(MS3).The lowest detection level of the cytokinins of the method reaches 0.1-2.0 pg with a very wide range of linear regression from 1-512 pg,at the coefficient factors of 0.98-0.99.The feasibility of this method has been proven in the application of the method to the analysis of the trace-amount contents of cytokinins in the micro-scale samples of various types of plant materials,such as aerial parts of rice and poplar leaves etc.12 endogenous cytokinins had been identified and quantified in the plant tissues,with an acceptable relatively higher recovery rate from 40% to 70%.     

Comprehensive separation and identification of chemical constituents from Apocynum venetum leaves by high-performance counter-current chromatography and high performance liquid chromatography coupled with mass spectrometry

High-performance counter-current chromatography (HPCCC) and high performance liquid chromatography coupled with mass spectrometry (HPLC-MS) was efficiently utilized for the separation and identification of the chemical components with a wide range of polarity from the mixed extract of Chinese medicinal herb Apocynum venetum. For HPCCC separation, four sets of solvent systems, n-hexane-ethyl acetate-acetonitrile-water (1.5:3.5:2:4.5, v:v:v:v), ethyl acetate-methanol-water (5:2:5, v:v:v) and n-butanol-methanol-water (5:1:5, v:v:v) were used for the one-step separation by four stages. The HPCCC separation was initiated by filling the column with the lower phase of n-hexane-ethyl acetate-acetonitrile-water (1.5:3.5:2:5, v:v:v:v) as a stationary phase followed by elution with the upper phase of n-hexane-ethyl acetate-acetonitrile-water (1.5:3.5:2:5, v:v:v:v) to separate the hydrophobic compounds (tail to head). Then the mobile phase was switched to the upper phase of ethyl acetate-acetonitrile-water (5:3:7, v:v:v) to eluted the moderate hydrophobic compounds, then the mobile phase was switched to the upper phase of ethyl acetate-methanol-water (5:2:5, v:v:v) to eluted the moderate hydrophilic compounds, and finally the hydrophilic compounds still retained in the column was eluted by the upper phase of n-butanol-methanol-water (5:1:5, v:v:v). A total of 16 named compounds including adhyperforin, hyperforin, amentoflavone, biapigenin, quercetin, avicularin, acetylated isoquercetin, acetylated hyperoside, astragalin, trifolin, isoquercetin, hyperoside, querciturone, rutin, chlorogenic acid and quercetin-3-O-β-D-glucosyl-β-D-glucopyranoside were successfully separated via the four sets of solvent systems in one step operation for 130 min. The compounds separated by HPCCC were identified by comparing with mixed standards data of HPLC-MS as well as NMR data.     

High-performance liquid chromatography with atmospheric pressure chemical ionization and electrospray ionization mass spectrometry for analysis of Angelica sinensis

An HPLC-PAD-API/MS method for analysing the chemical constituents of Angelica sinensis (A. sinensis) has been developed. ESI and APCI spectra, in both positive ion (PI) and negative ion (NI) modes, provided very useful information concerning the molecular weights of detected compounds. By comparing the retention times, UV spectra, mass spectra and molecular weights of detected compounds with those published in literature, 15 constituents of A. sinensis could be tentatively identified. This technique involving combined MS information may provide an objective, reliable and rapid analytical method for the quality control and database research of traditional Chinese medicines.     

Quantitative analysis of human serum corticosterone by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry

An original method based upon high-performance liquid chromatography coupled to electrospray ionization mass spectrometry has been developed for corticosterone (B) quantification in human serum. After extraction by diethyl ether using triamcinolone (T) as an internal standard, solutes are separated on a C₁₈ microbore column (250×1.0 mm, I.D.), using acetonitrile–water–formic acid (40:59.9:0.1, v/v/v) as the mobile phase (flow-rate 40 μl/min). Detection is performed on an API 1 single quadrupole mass spectrometer equipped with a ESI interface and operated in positive ionization mode. Corticosterone quantifications were realized by computing peak area ratios (B/T) of the serum extracts analyzed in SIM mode (m/z 347 and m/z 395 for B and T, respectively), and comparing them with the calibration curve (r=0.998).     

Ammonia desorption chemical ionization mass spectrometry of phospholipids

Ammonia desorption chemical ionization mass spectra (NH₃-DCIMS) of phosphatidyl-sulfocholines, a mixture of a homologous series of phosphatidylsulfocholines (PSC) and a mixture of a PSC phosphatidylcholine (PC), were measured by a flash heating method involving introduction of 1 μg of the samples on a tungsten wire, quickly heated, into the ion source of a Finnigan 4000/INCOS quadrupole instrument. It was possible to observe mass spectra during several seconds.The first spectra recorded of the PSCs gave essentially only the quasi-molecular [M + 18]⁺ peaks and the ‘A’ (see text) peaks. Spectra of a mixture of three homologous PSCs clearly showed three pairs of the above peaks. In contrast spectra of the PCs gave principally the [M + 1]⁺ and the ‘A’ peaks after 2s so that one can distinguish the diagnostic peaks in a mixture of a PC and a PSC.These results show that ammonia desorption chemical ionization by fast heating is a suitable technique for the charaterization of some head groups of phospholipids as well as a promising method for the analysis of phospholipid mixtures.     

Analysis of aminophospholipid molecular species by high performance liquid chromatography

A new method is described for the separation of individual molecular species of the aminophospholipids, phosphatidylethanolamine and phosphatidylserine. Trinitrobenzene-sulfonic acid was used to derivatize both aminophospholipids and the derivatives were purified by thin-layer chromatography. A reversed-phase high performance liquid chromatography technique was developed to separate and quantify individual molecular species based upon ultraviolet detection of the attached chromophore. The retention times of the molecular species on the C18 reversed-phase column were longer with increasing carbon chain length and decreasing degree of unsaturation of fatty acyl chain. The overall procedure allowed a quantitative recovery of the aminophospholipid species. The lower limit of detection was about 10 pmol and a linear response was observed in the range of 0.1-10 nmol of phospholipid. Using this method, we were able to separate and quantify trinitrophenyl-phosphatidylethanolamine molecular species of both subclasses (diacyl and alkenyl) from human red blood cells and rat brains. Separation of species was confirmed by gas-liquid chromatographic analysis of the fatty acid content of each peak and by thermospray liquid chromatography-mass spectrometry. This new method provides a convenient and sensitive technique for studies of aminophospholipid molecular species composition. Furthermore, it appears to be a useful tool for the analysis of asymmetric distribution of these species in biological membranes.     

Analysis of phospholipid species in human blood using normal-phase liquid chromatography coupled with electrospray ionization ion-trap tandem mass spectrometry

A narrow-bore normal-phase high-performance liquid chromatography (HPLC) method was developed for separation of phospholipid classes in human blood. The separation was obtained using an HPLC diol column and a gradient of chloroform and methanol with 0.1% formic acid, titrated to pH 5.3 with ammonia and added 0.05% triethylamine. The HPLC system was coupled on-line with an electrospray ionisation ion-trap mass spectrometer. Chromatographic baseline separation was obtained between phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, lyso-phosphatidylcholine, phosphatidylinositol and phosphatidylserine, eluting in that order. The total run time was 30 min. Plasmalogen phosphatidylethanolamine and sphingomyelin, which both are substances with structural similarities to the glycerophospholipids, had similar retention time as phosphatidylethanolamine, but were well separated from the other glycerophospholipid classes. The species from each class were identified using MS² or MS³, which forms characteristic lyso-fragments. The combination of lyso-fragment mass, molecular ion and chromatographic retention time was used to identify each species, including 20 species of phosphatidylglycerol. The mass spectra obtained for the phospholipid classes are presented. Using this system 17 disaturated phospholipid species not earlier described to be present in blood were identified. The limit of detection varied between different phospholipid classes and was in the range 0.1–5 ng of injected substance.     

Quantitative analysis of levamisole in porcine tissues by high-performance liquid chromatography combined with atmospheric pressure chemical ionization mass spectrometry

This work presents the development and the validation of an LC–MS–MS method with atmospheric pressure chemical ionization for the quantitative determination of levamisole, an anthelmintic for veterinary use, in porcine tissue samples. A liquid–liquid back extraction procedure using hexane–isoamylalcohol (95:5, v/v) as extraction solvent was followed by a solid-phase extraction procedure using an SCX column to clean up the tissue samples. Methyllevamisole was used as the internal standard. Chromatographic separation was achieved on a LiChrospher^® 60 RP-select B (5 μm) column using a mixture of 0.1 M ammonium acetate in water and acetonitrile as the mobile phase. The mass spectrometer was operated in MS–MS full scanning mode. The method was validated for the analysis of various porcine tissues: muscle, kidney, liver, fat and skin plus fat, according to the requirements defined by the European Community. Calibration graphs were prepared for all tissues and good linearity was achieved over the concentration ranges tested (r>0.99 and goodness of fit <10%). Limits of quantification of 5.0 ng/g were obtained for the analysis of levamisole in muscle, kidney, fat and skin plus fat tissues, and of 50.0 ng/g for liver analysis, which correspond in all cases to half the MRLs (maximum residue limits). Limits of detection ranged between 2 and 4 ng/g tissue. The within-day and between-day precisions (RSD, %) and the results for accuracy fell within the ranges specified. The method has been successfully used for the quantitative determination of levamisole in tissue samples from pigs medicated via drinking water. Moreover the product ion spectra of the levamisole peak in spiked and incurred tissue samples were in close agreement (based on ion ratio measurements) with those of standard solutions, indicating the worthiness of the described method for pure qualitative purposes.     

Determination of ritodrine in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A simple and sensitive HPLC/MS/MS method was developed and evaluated to determine the concentration of ritodrine (RTD) in human plasma. Liquid-liquid extraction with ethyl acetate was employed as the sample preparation method. The structural analogue salbutamol was selected as the internal standard (IS). The liquid chromatography was performed on a Hanbon Sci. & Tech. Lichrospher CN (150 mm x 4.6 mm, i.d., 5 microm) column (Hanbon, China) at 20 degrees C. A mixture of 0.03% acetic acid and methanol (50:50, v/v) was used as isocratic mobile phase to give the retention time 3.60 min for ritodrine and 2.94 min for salbutamol. Selected reaction monitoring (SRM) in positive ionization mode was employed for mass detection. The calibration functions were linear over the concentration range 0.39-100 ng mL(-1). The intra- and inter-day precision of the method were less than 15%. The lower limit of quantification was 0.39 ng mL(-1). The method had been found to be suitable for application to a pharmacokinetic study after oral administration of 20mg ritodrine hydrochloride tablet to 18 healthy female volunteers. The half-life is 2.54+/-0.67 h.     

Metallothionein separation and analysis by reversed phase high performance liquid chromatography coupled with graphite furnace atomic absorption spectrometry

Abstract

The feasibility of using reversed-phase high performance liquid chromatography (RP-HPLC) for the separation of metallothioneins (MTs) and subsequent determination of cadmium in MTs by graphite furnace atomic absorption spectrometry (GFAAS) in rabbit kidney and liver has been studied. RP-HPLC was used to isolate, characterise and quantitate liver and kidney MT isoforms. The MTs were eluted from a radially compressed C18 column with a neutral sodium phosphate buffer and detected by UV absorbance at 254 nm. Rabbit liver MTs was found to be comprised of seven distinct isoforms with five of which were found to be subspecies of the MT-I isoform. Rabbit kidney MTs exhibited only two predominant isoforms. A standard calibration curve was constructed using purified rabbit kidney MT-I and MT-II which demonstrated excellent linear correlation between peak height and the quantity of MT injected into the column. Recovery of MT from RP-HPLC was found to exceed 90%. Kidney and liver tissues from rabbit by feeding low levels of cadmium in diets was assayed using the RP-HPLC analysis of cytosol samples. Feeding stable cadmium in the diet resulted in the deposition of MT in the kidney rather than in the liver. The cadmium content in MT isoforms was determined by GFAAS. Less than 10% of the total cadmium in kidney was associated with MTs.     

Determination of tamsulosin in dog plasma by liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry

A rapid, sensitive and accurate liquid chromatographic-tandem mass spectrometric method is described for the determination of tamsulosin in dog plasma. Tamsulosin was extracted from plasma using a mixture of hexane-ethyl acetate (2:1, v/v) and separated on a C18 column interfaced with a triple quadrupole tandem mass spectrometer. The mobile phase consisting of a mixture of methanol, water and formic acid (80:20:1, v/v/v) was delivered at a flow rate of 0.5 ml/min. Atmospheric pressure chemical ionization (APCI) source was operated in positive ion mode. Selected reaction monitoring (SRM) mode using the transitions of m/z 409-->m/z 228 and m/z 256-->m/z 166.9 were used to quantify tamsulosin and the internal standard, respectively. The linearity was obtained over the concentration range of 0.1-50.0 ng/ml for tamsulosin and the lower limit of quantitation was 0.1 ng/ml. For each level of QC samples, inter- and intra-run precision was less than 5.0 and 4.0% (relative standard deviation (R.S.D.)), respectively, and accuracy was within +/-0.3% (relative error (R.E.)). This method was successfully applied to pharmacokinetic study of a tamsulosin formulation product after oral administration to beagle dogs.     

Chloride attachment negative-ion mass spectra of sugars by combined liquid chromatography and atmospheric pressure chemical ionization mass spectrometry

A method has been developed for the rapid determination of sugars, including molecular weight measurements, using high-performance liquid chromatography coupled with negative-ion, atmospheric-pressure chemical-ionization mass spectrometry. The chromatography was carried out on a 250 × 4 mm I.D. column packed with 7 μm NH₂-silica. The mobile phase consisted of a high percentage of methanol or acetonitrile with a small amount of chloroform. During the mass spectrometry, a strong base is formed from the polar solvent molecules by corona discharge, followed by ion—molecule reactions in the chemical ionization ion source (e.g. the methoxy anion is formed from methanol). This strong base reacts with the chloroform, generating chloride ions, which in turn react with the neutral sugar molecules as they elute from the chromatograph. The chloride ion and sugar interactions yield chloride-attachment ions, which are further stabilized by successive collisions. In this method, authentic monosaccharides and some oligosaccharides show dominant quasi-molecular ions, [M + Cl]⁻, with little fragmentation, and it is particularly useful for the molecular weight determination of sugars.     

Amino acid analysis by hydrophilic interaction chromatography coupled on-line to electrospray ionization mass spectrometry

Summary. Collagens form a common family of triple-helical proteins classified in 21 types. This unique structure is further stabilized by specific hydroxylation of distinct lysyl and prolyl residues forming 5-hydroxylysine and hydroxyproline (Hyp) isomers, mostly 4-trans and 3-trans-Hyp. The molecular distribution of the Hyp-isomers among the different collagen types is still not well investigated, even though disturbances in the hydroxylation of collagens are likely to be involved in several diseases such as osteoporosis and autoimmune diseases. Here, a new approach to analyze underivatized amino acids by hydrophilic interaction chromatography (HILIC) coupled on-line to electrospray ionization mass spectrometry (ESI-MS) is reported. This method can separate all three studied Hyp-isomers, Ile, and Leu, which are all isobaric, allowing a direct qualitative and quantitative analysis of collagen hydrolysates. The sensitivity and specificity was increased by a neutral loss scan based on the loss of formic acid (46 u).     

Quantitative analysis of heterocyclic amines in urine by liquid chromatography coupled with tandem mass spectrometry

A sensitive, reproducible, and rapid analytical method for the analysis of trace-level heterocyclic amines (HCAs) that are expected to have high levels of human exposure was developed. Liquid–liquid extraction (LLE) with dichloromethane (DCM) followed by solid-phase extraction (SPE) was carried out. Liquid extraction with DCM under basic conditions was efficient in extracting HCAs from urine samples. For further purification, mixed mode cationic exchange (MCX) cartridges were applied to eliminate the remaining interferences after liquid extraction. Separation and quantification were performed by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) in selected reaction monitoring (SRM) mode. The overall recoveries ranged between 71.0% and 113.6% with relative standard deviations (RSDs) of 5.1% to 14.7% for the entire procedure. The limits of detection (LODs) and limits of quantification (LOQs) of the proposed analytical method were in the ranges of 0.04 to 0.10 ng/ml and 0.15 to 0.36 ng/ml, respectively. This method was applied to the analysis of monitoring in urine samples for Korean school children, and the results demonstrated that the method can be used for the trace determination of HCAs in urine samples.     

Quantification of ceramide species in biological samples by liquid chromatography electrospray ionization tandem mass spectrometry

We present an optimized and validated liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for the simultaneous measurement of concentrations of different ceramide species in biological samples. The method of analysis of tissue samples is based on Bligh and Dyer extraction, reverse-phase high-performance liquid chromatography separation, and multiple reaction monitoring of ceramides. Preparation of plasma samples also requires isolation of sphingolipids by silica gel column chromatography prior to LC-ESI-MS/MS analysis. The limits of quantification were in a range of 0.01-0.50 ng/ml for distinct ceramides. The method was reliable for inter- and intraassay precision, accuracy, and linearity. Recoveries of ceramide subspecies from human plasma, rat liver, and muscle tissue were 78 to 91%, 70 to 99%, and 71 to 95%, respectively. The separation and quantification of several endogenous long-chain and very-long-chain ceramides using two nonphysiological odd chain ceramide (C17 and C25) internal standards was achieved within a single 21-min chromatographic run. The technique was applied to quantify distinct ceramide species in different rat tissues (muscle, liver, and heart) and in human plasma. Using this analytical technique, we demonstrated that a clinical exercise training intervention reduces the levels of ceramides in plasma of obese adults. This technique could be extended for quantification of other ceramides and sphingolipids with no significant modification.     

Quantitative measurement of 3'-oxolutein from human retina by normal-phase high-performance liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry

3-Hydroxy-beta,epsilon-carotene-3'-one (3'-oxolutein) is the major oxidative metabolite of dietary carotenoids in the retina of the human eye. Elucidating the biochemical mechanism of its formation may provide helpful insight into the pathogenesis of age-related macular degeneration; however, it is found in relatively low quantities that require highly sensitive methods for quantitation from individual retinas. Normal-phase high-performance liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry allowed us to do quantitative analysis of 3'-oxolutein from central and peripheral retinas obtained from individual human donors. The limit of quantification for 3'-oxolutein in human retina at a signal-to-noise ratio of 10 was 6 pg. The precision of the assay yielded a coefficient of variation ranging from 4.7 to 7.4% and accuracies of 106-108%. A statistically significant (R = 0.99, p < or = 0.001) linear working range was achieved between 5 and 7200 pg. The 3'-oxolutein contents from 8-mm punches of the central macula and peripheral retina were found to be 375+/-192 and 191+/-95 pg/tissue, respectively.     

Determination of imipramine in plasma by high pressure liquid chromatography and field ionization mass spectrometry: increased sensitivity in comparison with gas chromatography mass spectrometry

A quantitative method is reported for the determination of imipramine in plasma samples in the low nanogram and subnanogram range. The sensitivity and precision of the technique, which involves high pressure liquid chromatography and direct probe field ionization mass spectrometry, are approximately an order of magnitude greater than are offered by gas chromatography mass spectrometry with selected ion monitoring using deuterated or other types of internal standards. [2H6]Imipramine, labeled in the ethylene bridge and in the aromatic rings, serves as the isotopic diluent. The method has been used for the determination of the comparative bioavailabilities of two different commercial preparations of imipramine. In these tests, subjects ingested a 25 mg tablet of one or the other drug preparation together with a solution containing an equivalent amount of imipramine deuterated in the ethylene bridge ([2H2]imipramine). The latter served as an internal check for intrasubject variability in absorption of the imipramine tablets. Typical results from one of the subjects are presented.     

Characterization of flavonoids in the traditional Chinese herbal medicine-Huangqin by liquid chromatography coupled with electrospray ionization mass spectrometry

The root of Scutellaria baicalensis, called Huangqin in Chinese, is one of the most commonly used traditional Chinese medicines for the treatment of hepatitis, tumors, diarrhea, and inflammatory diseases. The major chemical constituents of Huangqin are flavonoids. In the present paper, HPLC-DAD-ESI-MS(n) was used to analyze flavonoids in the roots of S. baicalensis. A total of 26 flavonoids were identified or tentatively characterized, including 5 C-glycosides, 12 O-glycosides, and 9 free aglycones. Two C-glycosides, apigenin-6-C-glucyl-8-C-arabinoside and chrysin-6,8-di-C-glucoside, together with some O-glycosides, are reported from S. baicalensis for the first time. This method is simple, reliable and sensitive, and could be used for the quality control of Huangqin and its related preparations.