High-performance liquid chromatography of proteins

Incubation of liver microsomes with GDP [14C] mannose leads to the formation of lipid-linked derivatives of [14C] mannose, a dolichol phosphate monosaccharide and dolichol pyrophosphate oligosaccharides. Standard procedures for separating these two types of compounds from each other were found to be deficient in that fractions thought to contain only dolichol pyrophosphate oligosaccharides are contaminated with dolichol phosphate mannose. This paper presents a column chromatographic procedure which conveniently separates the products of an 8 min labeling experiment into two components; dolichol phosphate [14C]mannose and a [14C]-mannose containing oligosaccharide which is also lipid bound. When this oligosaccharide is released from the lipid by hydrolysis and chromatographed on Sephadex G-50 or G-15 it gives a single peak with an indicated molecular weight of 1100. However, when this released oligosaccharide is chromatographed on concanavalin A Sepharose it is resolved into two peaks suggesting that there may be 2 oligosaccharide of approximately the same size but different structures. After brief periods of labeling with GDP [14C]mannose (5 s) an additional oligosaccharide of 3 to 4 sugar residues can be found in the dolichol pyrophosphate oligosaccharides fraction. Incubation of liver microsomes with UDP [14C]glucose or UDP[14C]galactose produces oligosaccharide components containing 7--8 sugar residues. Labeling of microsomes with UDP[14C]acetylglucosamine gives rise to three different components, including a lipid bound oligosaccharide containing 3- 5 sugar residues.     

Determination of provitamin A of green leafy vegetables by high performance liquid chromatography and open column chromatography

AIM OF STUDY: Intrathecal sufentanil has recently been used in labour as part of a combined spinal epidural technique. This study was conducted to compare its use in combination with bupivacaine for caesarean section with fentanyl added to bupivacaine and bupivacaine alone. METHODS: Sixty ASA I and II patients for non-emergency caesarean section under spinal anaesthesia were divided into three groups to receive 15 micrograms fentanyl added to 7.5 mg bupivacaine, 10 micrograms sufentanil added to 7.5 mg bupivacaine and 7.5 mg bupivacaine. Onset time of sensory blockade, side effects, surgical conditions, neonatal outcome and quality of the anaesthetic was assessed. On the first postoperative day, duration of effective analgesia, side effects and patient satisfaction were noted. RESULTS: The duration of effective analgesia of bupivacaine alone was prolonged with the addition of sufentanil and fentanyl by 358% and 256% respectively. No patient in the sufentanil and fentanyl groups required additional intra-operative analgesics compared with 17.6% of patients in the bupivacaine alone group. There was an increase in incidence of desaturation in the sufentanil group (45%) and fentanyl group (5.6%) compared with the bupivacaine only group (0%). The incidence of pruritus was 35% with sufentanil, 27.8% with fentanyl against 0% with bupivacaine alone. CONCLUSION: The addition of 10 micrograms of sufentanil and 15 micrograms of fentanyl to 7.5 mg of bupivacaine prolonged the duration of effective analgesia and improved intra-operative analgesia. However, the incidence of pruritus and episodes of desaturation were increased more with 10 micrograms sufentanil than with 15 micrograms fentanyl.     

High-performance liquid chromatography determination of mycophenolic acid and its glucuronide metabolite in human plasma

Two HPLC-UV assays are reported here: one is a rapid assay for mycophenolic acid (MPA) and the other is a simultaneous assay for MPA and its metabolite mycophenolic acid glucuronide (MPAG). For both methods, plasma samples (500 microl) with added internal standard were acidified and extracted using C18 solid-phase extraction cartridges. Chromatographic separation was achieved on a C18 Novapak column using a mobile phase consisting of methanol-0.05% orthophosphoric acid (40:60, v/v) for the rapid MPA assay and 30:70 for the simultaneous MPA and MPAG assay. The assays were linear over the ranges 0.1 to 50.0 mg/l for MPA and 2.8 to 225.8 mg/l for MPAG. Mean absolute recovery for all analytes was >99%. These methods are suitable for therapeutic drug monitoring and pharmacokinetic studies.     

High-performance liquid chromatographic determination of tramadol in human plasma

Tramadol has been determined in human plasma samples using a sensitive high-performance liquid chromatographic method. The plasma samples were extracted with tert.-butylmethyl ether in one-step liquid-liquid extraction (recovery 86%) and analyses of the extracts were performed on reversed-phase silica gel using ion-pair chromatography (verapamil as an internal standard) and fluorescence detection. The method was applied to the determination of tramadol levels in twelve healthy volunteers after oral administration of 100 mg of tramadol in capsules of Protradon and Tramal.     

High-performance liquid chromatography of penimocycline (author's transl)

Penimocycline is an antibiotic obtained by Mannich reaction between tetracycline and ampicilline. Separation of penimocycline from tetracyclines and other impurities has been studied by high-performance liquid chromatography. The most effective method is liquid-liquid partition on a Micropak CH column (non-polar hydrocarbon bonded on porous silica microparticles) and gradient elution with water-methanol, 0.02 M phosphate buffer (pH 7.6) and 1 mM EDTA. Some results on hydrolysis of penimocycline are given.     

High-performance liquid chromatographic assay for fenoprofen in human plasma

A high-performance liquid chromatographic method is described for the quantitation of fenoprofen, dl-2-(3-phenoxyphenyl)-propionic acid, in human plasma. The proteins in plasma were precipitated by the addition of hydrochloric acid. Fenoprofen and the internal standard, dl-2-(4-phenoxyphenyl)valeric acid, were extracted into butyl chloride and then back-extracted into sodium hydroxide. The aqueous solution was injected onto a reversed-phase alkylphenyl column, and the compounds were eluted using a mobile phase of acetonitrile-water-acetic acid (50:50:2 v/v/v). At a flow rate of 1 ml/min, the retention times of fenoprofen and the internal standard were 8 and 12 min, respectively. The absorbance was monitored at 272 nm. The method requires 1.0 ml of plasma and is sensitive to 0.5 microgram/ml. This procedure has been used for routine assay of multiple samples from bioavailability and compliance studies.     

Supercritical fluid extraction and reversed-phase liquid chromatography methods for vitamin A and beta-carotene heterogeneous distribution of vitamin A in the liver

In the present study, we have performed chemical investigations of the stem cell walls during internode maturation in order to study the growth dynamics of alfalfa and the deposition of the main cell wall components (polysaccharides and lignins). Internode cell walls were analysed by chemical fractionation using a mild delignification step aiming at sequential removal of polysaccharides and lignins. Delignification facilitated the subsequent removal of the xylose-rich polysaccharides by NaOH extraction as previously shown. This trend was more pronounced in the case of older internodes which have a larger proportion of secondary tissues containing syringyl type lignins in contrast to younger ones which are mainly composed of primary tissues containing guaiacyl type lignins and pectin rich cell walls.     

High-performance liquid chromatography of selected organic peroxides with oxidative amperometric detection

The ability of the flagellate Crithidia fasciculata to induce encystation of the reptile pathogen, Entamoeba invadens, was studied in vitro. A specific ratio of flagellate to amoeba was required; both live and heat-killed C. fasciculata induced amoebic encystation. The interaction between the Crithidia and Entamoeba cells was found to be galactose-mediated because the addition of galactose to the culture medium, or the pretreatment of the flagellate with galactosidase, eliminated its ability to induce encystation. Galactose was also found to prevent the amoeba amoeba aggregation that normally occurs in axenic cultures of encystation-induced E. invadens. Both galactose and glcNAc completely inhibited cyst formation of these induced cultures, although the latter sugar did not prevent cell aggregation. These results indicate that a galactose-mediated interaction between E. invadens cells is an early step in the in vitro encystation pathway.     

High-performance liquid chromatography separation and quantitation of methylprednisolone from rat brain

A sensitive, reliable method for the extraction, separation, and quantitation of methylprednisolone from rat brain is reported. The method can accurately quantitate methylprednisolone levels between 9.8 and 2500 ng/injection using a two-step HPLC separation and monitoring absorbance at 254 nm. A 90% extraction recovery of methylprednisolone (interday variation of 9.0% and an intraday variation of 0.0 to 7.7%) from rat cortex was obtained with a double extraction method using low toxicity solvents. These solvents are known to quantitatively extract the neutral lipids and phospholipids from brain. Combined with the ability to separate the neutral lipid and methylprednisolone fractions for further separation, and the ability to separate all phospholipid classes in the first run, this method offers great utility combined with the reliable, high extraction recovery and sensitive quantitation of methylprednisolone.     

High-performance liquid chromatographic determination of 4-methylpyrazole in plasma and in dialysate

A rapid method for the determination of 4-methylpyrazole (4-MP) levels in plasma and in dialysate by isocratic reversed-phase high-performance liquid chromatography with UV detection is described. The internal standard was the 3-methylpyrazole (3-MP). Plasma sample preparation consisted of a protein precipitation. Dialysate samples were injected without preparation. The method was linear up to 30 mg l(-1) in plasma and up to 5 mg l(-1) in dialysate. The within-day precisions (C.V.) were less than 4% in plasma and were less than 2% in dialysate. The day-to-day precisions (C.V.) were less than 7% in plasma and were less than 3% in dialysate. This method is easy to perform and has practical interest for clinicians who need to monitor in emergency 4-MP levels in ethylene glycol and methanol poisonings.     

High-performance liquid chromatographic method for measuring total plasma homocysteine levels

We have modified a high-performance liquid chromatographic (HPLC) procedure based on SBD-F (ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate) pre-column derivatization to obtain an assay that is useful for routine clinical total plasma homocysteine (tHcy) analysis. The introduction of easily handled sodium borohydride instead of the traditional tri-n-butylphosphine in dimethylformamide as a reductant and a 14-min run-time using basic isocratic HPLC equipment are the more notable advantages. The addition of mercaptopropionylglycine as an internal standard contributed to improvements in the reproducibility of the assay, yielding within- and between-run precisions of 1.9 and 4% (C.V.), respectively. Reference values for fasting tHcy were 7.65+/-2.3 and 8.9+/-2.4 micromol/l, while post-methionine load gave tHcy levels of 19.9+/-5.5 and 26.8+/-5.5 micromol/l, for women and men, respectively (n=40).     

High-performance liquid chromatography-mass spectrometry for the quantification of nortriptyline and 10-hydroxynortriptyline in plasma

A highly sensitive and selective method for the quantification of nortriptyline and its major 10-hydroxy metabolite in plasma is described. The method is based on liquid-liquid extraction in combination with acid dehydration of the 10-hydroxy metabolite to the less polar 10,11-dehydronortriptyline. Deuterium labelled internal standards ([2H4]NT and [2H3]10-OH-NT) were used and the compounds were separated by reversed-phase HPLC and detected using atmospheric pressure chemical ionisation and mass spectrometry. The limit of quantification was 0.8 ng/ml for both compounds. A 1-ml volume of plasma was used for analysis in the concentration range 0.8-32 ng/ml. The within- and between-day coefficients of variation were 11% in the low, 1.6 ng/ml range, and 7% at 8 ng ml/ml. Using this method it was possible to quantify plasma concentrations for 168 h following a single oral dose of 25 mg of nortriptyline with good accuracy and precision.     

Analysis of prednisolone in plasma by high-performance liquid chromatography

A sensitive, specific high-performance liquid chromatographic procedure for the determination of prednisolone in plasma is described. The organic solvent extract from plasma is chromatographed on a silica gel column using a mobile phase of 0.2% glacial acetic acid, 6% ethanol, 30% methylene chloride in n-hexane on a high-performance liquid chromatograph fitted with an ultraviolet dector (254 nm). Quantitation of plasma samples containing 25 ng/ml prednisolone is reported. Metabolites and endogenous hydrocortisone do not interfere with prednisolone. The determination of prednisolone concentration in plasma following administration of a 10-mg single oral dose to a human subject is described.     

High-performance liquid chromatographic determination of diclofenac in human plasma using automated column switching

A validated high-performance liquid chromatographic procedure employing ultraviolet detection for the analysis of diclofenac in human plasma is reported. The method is rapid and, coupled with column switching, leads to a sensitive, accurate and reproducible assay. The retention times of diclofenac and the internal standard (4'-methoxydiclofenac, CGP-4287) are 6.4 and 7.6 min, respectively. The peak height versus plasma concentration is linear over the range 5.0-2000 ng/ml with a detection limit below 2.5 ng/ml. The mean absolute recovery of diclofenac using the described assay is 96.5% (n = 24). The inter- and intra-day accuracy and precision are within 8.3% of the actual values for all concentrations investigated. Furthermore, this procedure is applied to assess the pharmacokinetics of a single 75-mg oral dose of diclofenac sodium.     

Quantification of flumequine enantiomers in plasma by high-performance liquid chromatography

A 62-year-old woman with pulmonary hypertension underwent mitral valve re-replacement through right thoracotomy. Severe adhesion occurred to the right lung. During pleural dissection the lung collapsed under single-lung ventilation, rapidly elevated pulmonary vascular resistance caused hemodynamic instability. When pulmonary hypertension is preoperatively present, this approach under single-lung ventilation is not recommended.     

Determination of ivermectin in human plasma by high-performance liquid chromatography

A specific reversed-phase HPLC-assay with sensitive fluorometric detection has been developed to measure the potent new antiparasitic agent ivermectin (CAS 70288-86-7) in human plasma (and urine). The lower limit of the method was 1 ng/ml and the intra-/interassay variability averaged 4.5/6.9%, respectively. The assay was applied for measuring plasma (urine) concentrations of ivermectin upto 56 (72 h) following a single oral dose of 6 and 12 mg. No unchanged or conjugated ivermectin could be detected in urine. Plasma concentrations increased linearly with dose but elimination half-life (12.6/13.4 h) was independent of the administered dose. Thus, the method is applicable for monitoring plasma levels during clinical and pharmacokinetic trials with ivermectin to evaluate its most efficacious dosage regimen.     

Determination of sinefungin in rat plasma by high-performance liquid chromatography

This is the 31st article in a continuing series of objectives to direct emergency medicine resident experiences on off-service rotations. Neck and torso trauma accounts for a large portion of injuries, and its management is an essential part of training in emergency medicine. Due to the often life-threatening presentations of trauma victims, resident instruction may be conducted at the bedside in difficult and demanding situations. Therefore, it is essential for residents to have specific goals and objectives to guide their acquisition of knowledge required to make critical decisions for patients with major trauma.     

Assay of sulfonylureas in human plasma by high-performance liquid chromatography

A sensitive and specific high-performance liquid chromatographic procedure for the determination of chlorpropamide or tolbutamide in plasma in the presence of their metabolites is described. The ether extract of acidified plasma is redissolved in the mobile phase, 17% acetonitrile in 0.05 M aqueous ammonium formate, and chromatographed on a reverse-phase column on a high-performance liquid chromatograph fitted with a UV absorbance detector. Quantitation of plasma samples containing less than 0.5 mug/ml of chlorpropamide and 5 mug/ml of tolbutamide is reported, using these drugs as mutual internal standards. The retention times of the metabolites are such that they do not interfere in the procedure. The assay method was tested in a human volunteer with both drugs and found suitable for single-dose pharmacokinetic studies.