The identification of serum tuftsin by reverse-phase high-performance liquid chromatography and mass spectrometry

We describe for the first time a method for unambiguously identifying the phagocytic stimulating tetrapeptide tuftsin from trypsinized human serum. The method consists of separating serum tuftsin by reverse-phase (RP)-HPLC, collecting HPLC fractions corresponding to the synthetic tuftsin retention time, and then subjecting those fractions to mass spectrometry/mass spectrometry (MS/MS) analysis, which provides optimal molecular specificity to the measurement. Although quantification was not the goal, it was estimated that the amount of tuftsin found by RP-HPLC and MS/MS was in the hundreds of nanograms per milliliter.     

Reversed-phase high-performance liquid chromatographic screening method for serum steroids using retention index and diode-array detection

A multisteroid screening method has been developed based on the use of 1-[4-(2,3-dihydroxypropoxy)phenyl]-1-alkanones as retention index standards and UV absorbance spectra recorded on-line with a diode-array detector using reversed-phase high-performance liquid chromatographic gradient elution with acetonitrile and water. The effect of chromatographic conditions on retention indices of steroids were studied. The method was tentatively applied to profiling of steroids in serum samples.     

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).     

Differentiated quantification of human bile acids in serum by high-performance liquid chromatography-tandem mass spectrometry

Determination of quantitative changes in the pattern of serum bile acids is important for the monitoring of diseases affecting bile acid metabolism. A sensitive and specific high-performance liquid chromatography (HPLC)-MS/MS method was developed for the differentiated quantification of unconjugated as well as glycine- and taurine-conjugated cholic, chenodeoxycholic (CDCA), deoxycholic (DCA), ursodeoxycholic (UDCA) and lithocholic acid (LCA) in serum samples. After solid-phase extraction and reversed-phase HPLC separation, detection of the conjugated bile acids was performed using electrospray ionization (ESI)-MS/MS and selected reaction monitoring mode, whereas unconjugated bile acids were determined by ESI-MS and selected ion monitoring mode. The within-day and between-day coefficients of variation were below 7% for all bile acids and the recovery rates of the extraction procedure were between 84.9 and 105%. The developed method was applied to a group of 21 healthy volunteers and preliminary reference intervals in serum were established. In patients with drug-induced cholestasis, an elevation of primary bile acids has been shown.     

Reversed-phase high-performance liquid chromatographic assay for the adenovirus type 5 proteome

An RP-HPLC assay was developed for a recombinant adenovirus type 5. During chromatography, intact adenovirus dissociated into its structural components (DNA and proteins) and the viral proteome was separated yielding a characteristic fingerprint. The individual components were identified by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy, N-terminal sequencing and amino acid composition. The assay was utilized to measure adenovirus particle concentration through quantification of structural proteins. Each structural protein provided independent measurement of virus concentration allowing verification of accuracy. The assay sensitivity is at or below 2·10⁸ particles. Contrary to the benchmark spectrophotometric assay, the RP-HPLC assay was shown to be insensitive to contaminants common for partially purified adenovirus preparations.     

Simultaneous glycosylation analysis of human serum glycoproteins by high-performance liquid chromatography/tandem mass spectrometry

Changes in the glycosylation of some serum proteins are associated with certain diseases. In this study, we performed simultaneous site-specific glycosylation analysis of abundant serum glycoproteins by LC/Qq-TOF MS of human serum tryptic digest, the albumin of which was depleted. The glycopeptide peaks on the chromatogram were basically assigned by database searching with modified peak-list text files of MS/MS spectra and then based on mass differences of glycan units from characterized glycopeptides. Glycopeptide of IgG, haptoglobin and ceruloplasmin were confirmed by means of a comparison of their retention times and m/z values with those obtained by LC/MS of commercially available glycoproteins. Mass spectrometric carbohydrate heterogeneity in the assigned glycopeptides was analyzed by an additional LC/MS. We successfully demonstrated site-specific glycosylation of 23 sites in abundant serum glycoproteins.     

Reversed-phase high-performance liquid chromatographic analysis of thiamine phosphate esters at subpicomole levels

Separation and determination of thiamine phosphate esters were achieved by reversed-phase high-performance liquid chromatography (hplc) after conversion to corresponding thiochrome esters. The elution order was thiochrome triphosphate, thiochrome pyrophosphate, and thiochrome monophosphate by a system composed of 25 mm potassium phosphate buffer (pH 8.4) and 2.5% N,N-dimethylformamide. The minimum amount reproducibly detected was 0.05 pmol for each thiochrome phosphate. Thiamine phosphate esters in rat tissues were successfully determined by the reversed-phase hplc after alkaline oxidation of the tissue extract, which resulted in a good agreement in their contents to those obtained by the straight-phase hplc previously reported.     

high-performance liquid chromatography of amino acids, peptides and proteins : LXIII. Reversed-phase high-performance liquid chromatographic characterisation of several polypeptide and protein hormones

The chromatographic behaviour on alkylsilicas of a variety of hormonal proteins is described. Optimization of resolution and recovery of these protein hormones, which included porcine relaxins, human chorionic gonadotropin, human placental lactogen, pituitary derived growth hormone and adenohypophyseal glycoprotein hormones, was achieved by manipulation of both mobile and stationary phase parameters. With standard stainless-steel analytical columns (10–30 cm × 0.4 cm) packed with meso- or macro-porous n-alkylsilica supports these proteins can be readily fractionated at the semi-preparative level with separation times generally under 90 min using elution systems directly compatible with subsequent methods of primary structure determination or biological functional analysis. The effects of changes in several experimental parameters on peak symmetry, retention and recovery are described.     

Rapid high-performance liquid chromatographic determination of serum gabapentin

Gabapentin (GBP) is a new antiepileptic drug approved for clinical treatment of partial seizures in the USA. Serum GBP concentrations in 283 patients were studied using high-performance liquid chromatography with fluorescence detection. The standard curves were linear over a range of 60 ng to 15 μg/ml. The coefficient of variations were 3.4 to 8.8% and 1.4 to 9.8% for intra- and inter-assay studies, respectively. The lower limit of quantitation was 10 ng/ml. Of the 283 patients studied, 72.5% had GBP levels between 2 and 10 μg/ml, 14.8% were below 2 μg/ml and 12.7% above 10 μg/ml. The mean±S.E. of GBP in 283 patients was 5.38±0.23 μg/ml. Peak concentrations of more than 15 μg/ml and trough levels as low as 0.1 μg/ml were not uncommon. The method described was rapid, simple, highly sensitive and reproducible. Other antiepileptic drugs and endogenous compounds did not interfere with the assay.     

Rapid high-performance liquid chromatographic assay for total homocysteine levels in human serum

A rapid, isocratic high-performance liquid chromatographic (HPLC) method is described for the determination of total homocysteine levels in human serum. Prior to reversed-phase HPLC analysis, the serum thiols were derivatized with SBD-F (ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate), a thiol-specific fluorogenic probe which is commercially available. Retention of SBD-homocysteine was sensitive to pH, and a mobile phase pH of 2.1 ensured baseline separation of serum thiols within 6 min. The method is simple, sensitive, reproducible (between-run coefficient of variation of 6.6%) and very suitable for routine determination of serum homocysteine levels in a clinical pathology laboratory.     

Determination of proteins in infant formula by high-performance liquid chromatography-electrospray tandem mass spectrometry

To determine the protein content of formula, gel electrophoresis was performed on the infant formula samples and the entire protein patterns were analyzed by nano-high performance liquid chromatography-electrospray tandem mass spectrometry (nano-HPLC/ESI/MS/MS). From the commercial infant formula profiled in this study, a total of 154 peptides, corresponding to 31 unique proteins were identified by nano-HPLC/ESI/MS/MS. Each of the identified peptides was reconfirmed by a strict integrated approach using tandem mass spectra. This protein profiling method using gel electrophoresis coupled with nano-HPLC/ESI/MS/MS and manual evaluation is a sensitive and accurate method for protein identification as well as a powerful tool for monitoring various types of food products.     

Felodipine quantification in human plasma by high-performance liquid chromatography coupled to tandem mass spectrometry

A rapid, sensitive, robust and specific method was developed for the determination and quantitation of felodipine, in human blood plasma by liquid chromatography coupled with tandem mass spectrometry using nimodipine as internal standard. Felodipine was extracted from 0.5 mL human plasma by use of a liquid/liquid procedure using diethyl ether/hexane (80/20, v/v) as eluent. The method included a chromatographic run of 5 min using a C(18) analytical column (100 mm x 4.6 mm i.d.) and the calibration curve was linear over the range from 0.02 to 10 ng mL(-1) (r(2) > 0.994). The between-run precision, determined as relative standard deviation of replicate quality controls, was 5.7% (0.06 ng mL(-1)), 7.1% (0.6 ng mL(-1)) and 6.8% (7.5 ng mL(-1)). The between-run accuracy was +/- 0.0, 2.1 and 3.1% for the above-mentioned concentrations, respectively.     

Determination of ketoconazole in human plasma by high-performance liquid chromatography-tandem mass spectrometry

A simple, rapid and specific high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) has been developed and validated for the determination of ketoconazole in human plasma. The method used diethyl ether to extract the ketoconazole and the internal standard (I.S.) R51012 from alkalinized plasma sample. The LC separation was on a C(18) column (50 x 3 mm, 5 microm) using acetonitrile-water-formic acid (75:25:1, v/v/v) mobile phase. The retention times were approximately 1.8 min for both ketoconazole and the I.S. The MS-MS detection was by monitoring 531.2-->82.1 (m/z) for ketoconazole, and 733.5-->460.2 (m/z) for the I.S. The dynamic range was from 20.0 to 10000 ng/ml based on 0.1 ml plasma, with linear correlation coefficient of > or =0.9985. The run time was 2.5 min/injection. The recoveries of ketoconazole and the I.S. were 102 and 106%, respectively. The precision and accuracy of the control samples were with the relative standard deviations (RSDs) of < or =4.4% (n=6) and the relative errors (REs) from -0.6 to 1.4% for intra-day assay, and < or =8.6% RSD (n=18) and -1.4 to 0.9% RE for inter-day assay. The partial volume tests demonstrated good dilution integrity. Three freeze-thaw cycles, keeping plasma samples at ambient for 24 h, storing extracted samples at ambient for 24 h, and storing frozen plasma samples at approximately -20 degrees C for up to 2 months did not show substantial effects.     

Quantitation of iptakalim in human plasma by high-performance liquid chromatography-tandem mass spectrometry

This paper describes a rapid and sensitive analytical method for the quantitation of iptakalim, a novel antihypertensive drug, in human plasma. The method is based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) using sildenafil as internal standard. Sample preparation involved liquid-liquid extraction with dichloromethane-diethyl ether (2:3, v/v) in a basic environment. Chromatography was carried out on an amino column with a mobile phase consisting of acetonitrile-water (55:45, v/v, water containing 0.5% formic acid). Detection employed electrospray ionization (ESI) tandem mass spectrometry in the multiple-reaction-monitoring (MRM) mode. The assay was linear in the concentration range of 0.5-100 ng/ml with a lower limit of quantitation (LLOQ) of 0.5 ng/ml. Intra- and inter-day precision (R.S.D.) were <4.5% and <12.0%, respectively and the accuracy (R.E.) was in the range +/-5%. The method was successfully applied to a single oral dose pharmacokinetic study in human volunteers.     

Large-scale identification of Caenorhabditis elegans proteins by multidimensional liquid chromatography-tandem mass spectrometry

A proteome of a model organism, Caenorhabditis elegans, was analyzed by an integrated liquid chromatography (LC)-based protein identification system, which was constructed by microscale two-dimensional liquid chromatography (2DLC) coupled with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) on a high-resolution hybrid mass spectrometer with an automated data analysis system. Soluble and insoluble protein fractions were prepared from a mixed growth phase culture of the worm C. elegans, digested with trypsin, and fractionated separately on the 2DLC system. The separated peptides were directly analyzed by on-line ESI-MS/MS in a data-dependent mode, and the resultant spectral data were automatically processed to search a genome sequence database, wormpep 66, for protein identification. The total number of proteins of the composite proteome identified in this method was 1,616, including 110 secreted/targeted proteins and 242 transmembrane proteins. The codon adaptation indices of the identified proteins suggested that the system could identify proteins of relatively low abundance, which are difficult to identify by conventional 2D-gel electrophoresis (GE) followed by an offline mass spectrometric analysis such as peptide mass fingerprinting. Among the approximately 5,400 peptides assigned in this study, many peptides with post-translational modifications, such as N-terminal acetylation and phosphorylation, were detected. This expression profile of C. elegans, containing 571 hypothetical gene products, will serve as the basic data of a major proteome set expressed in the worm.