An automated alkaline elution system: DNA damage induced by 1,2-dibromo-3-chloropropane in vivo and in vitro

An automated alkaline elution system for the detection of DNA damage has been developed. After manual application of samples, which is completed within 5 min, the subsequent supply of liquids, changes in flow rates, and temperature are controlled automatically. The system operates 16 filters and may easily be expanded. The sensitivity of the fluorometric DNA determinations with the Hoechst 33258 dye is increased by using an elution buffer (20 mM Na2EDTA, pH 12.50) with low background fluorescence. DNA is determined using an automated setup similar to the one recently presented by Sterzel et al. (1985, Anal. Biochem. 147, 462-467). The most significant modification is the use of a neutralization buffer which allows variations in the pH of eluted fractions. This change increases the sensitivity of the DNA measurements. The automated alkaline elution system was evaluated using the nematocide 1,2-dibromo-3-chloropropane (DBCP) in a study of its genotoxic effects in the testes and the kidneys. Significant DNA damage was induced in testicular cells by 2.5 microM DBCP (1 h) in vitro and 85 mumol/kg DBCP ip (3 h) in vivo. The damage appeared after short treatment times (10 min in vivo). Variations in the observed DBCP response in vivo were largely due to interanimal variations. The automated alkaline elution system proved to be a sensitive assay also for the detection of DNA damage in kidney nuclei prepared from rats exposed to DBCP. Provided that kidney nuclei from untreated rats, mice, or hamster were kept ice-cold until lysing, 85-100% of their DNA was retained after 16 h of elution, indicating highly intact DNA. Under the same conditions, guinea pig DNA was rapidly degraded unless the nuclei were prepared in a buffer with a higher concentration of Na2EDTA (20 mM).     

Purification of VP3 protein of infectious bursal disease virus using nickel ion-immobilized regenerated cellulose-based membranes

In this study, hexa-histidine tagged VP3 protein of infectious bursal disease virus (IBDV) was purified using immobilized metal ion affinity technique from the fermentation of Escherichia coli BL21 (DE3) containing a recombinant plasmid with a VP3 gene. The purification efficiencies of VP3 protein (TVP3 and DeltaTVP3) using Ni(2+)-NTA commercial agarose gels and Ni(2+)-IDA regenerated cellulose-based membranes at 4 degrees C were compared. A good washing condition for removing most impurity proteins was found as 20 mM NaH(2)PO(4), 500 mM NaCl, 40 mM imidazole, pH 7.8, whereas an efficient elution condition was 20 mM NaH(2)PO(4), 500 mM NaCl, 500 or 750 mM imidazole, pH 7.8. By applying these conditions to the flow experiments, similar recovery (86-88%) and purity (98-99%) for VP3 were obtained in both gel column (1 ml gel) and membrane cartridge (four membrane disks) under the flow rate of 1.7 ml/min for protein loading and 2.7 ml/min for protein elution. Regarding that the membrane process exhibited some advantages such as shorter residence time and lower cost, a better process efficiency in a large-scale system could be expected for the Ni(2+)-IDA membranes.     

Measurement of DNA-protein crosslinks in mammalian cells without X-irradiation

To study the mechanisms of formation and repair of DNA-protein crosslinks in mammalian cells, the best general method to assay these lesions is the Kohn membrane alkaline elution procedure. Use of this sensitive technique requires the introduction of random strand breaks in the DNA by X-irradiation to reduce the very high molecular weight so that it elutes off the filter at an appropriate rate. This report describes an alternative method for fragmenting the DNA in the absence of X-irradiation equipment. Convenient reproducible elution rates of DNA from various mouse and human cells in culture without X-irradiation result from elution through polyvinyl chloride filters with 75 mM sodium hydroxide (0.33 ml/min) instead of the standard 20 mM EDTA-tetrapropylammonium hydroxide, pH 12.2 (0.03 to 0.04 ml/min). Dose-dependent retardation of the DNA elution was observed over the range 0 to 30 microM trans-platinum(II)diamminedichloride, and proteinase K treatment during cell lysis restored the elution rate to that of the untreated control cell DNA. In the absence of X-irradiation, this elution method measures DNA-protein crosslinks with higher sensitivity and equivalent reproducibility as the air-burst procedure.     

Comparison of polysaccharide-based and protein-based chiral liquid chromatography columns for enantioseparation of drugs

Two different columns—Lux Cellulose-1 and Chiralpak CBH—were evaluated for their chiral recognition abilities for eight drugs comprising three β-blockers, one antacid, and four cathinones in polar-organic elution mode and reversed-phase elution mode, respectively. The factors that affected the enantioseparation were tested and optimized to develop a suitable chiral separation method whose LC conditions are compatible with MS detection. In polar-organic elution mode with the Lux Cellulose-1 column, methanol and acetonitrile were tested as the main components of the mobile phase. In addition, the effects of adding isopropanol as organic modifier, acidic additives (formic acid), and basic additives (diethylamine) were evaluated. In reversed-phase elution mode with the Chiralpak CBH column, the effect of type and concentration of organic modifier (isopropanol, acetonitrile, and methanol), the mobile phase pH (6.4 and 5.0), and buffer concentration (1mM-20mM ammonium acetate) were evaluated. The best enantioseparation was achieved with the Chiralpak CBH column with a mobile phase composed of 5mM ammonium acetate aqueous (pH = 6.4)/methanol (95/5, v/v) at a flow rate of 0.1 mL/min and a temperature of 30°C. Under these conditions, six of eight chiral drugs were baseline separated.     

Genotoxic effects of paracetamol in V79 Chinese hamster cells

Paracetamol was studied for possible genotoxic effects in V79 Chinese hamster cells. Paracetamol (0.5 mM for 30 min) reduced the rate of DNA synthesis in exponentially growing V79 cells to about 50% of control. A further decrease in the DNA synthesis was seen during the first 30 min after termination of paracetamol exposure. Paracetamol (3 and 10 mM for 2 h) caused a small increase in DNA single-strand breaks, as measured by the alkaline elution technique. After 16 h elution, the amount of DNA retained on the filters was 79 and 70% of controls in cells treated with 3 and 10 mM paracetamol respectively. No indication of DNA damage was seen in measuring the effect of paracetamol (0.25-10 mM for 2 h) on unscheduled DNA synthesis in growth-arrested cultures of V79 cells. At the highest concentrations (3 and 10 mM paracetamol), decreased unscheduled DNA synthesis was observed. Also UV-induced DNA-repair synthesis was inhibited by 3 and 10 mM paracetamol. DNA-repair synthesis was, however, inhibited at a much higher concentration than that inhibiting replicative DNA synthesis. The number of sister-chromatid exchanges (SCE) increased in a dose-dependent manner on 2 h exposure to paracetamol from 1 mM to 10 mM. At the highest dose tested (10 mM), the number of SCE increased to 3 times the control value. Co-culturing the V79 cells with freshly isolated mouse hepatocytes had no further effect on the paracetamol induced sister-chromatid exchanges. The present study indicates that paracetamol may cause DNA damage in V79 cells without any external metabolic activation system added.     

Enzymatic synthesis of UDP-(3-deoxy-3-fluoro)-D-galactose and UDP-(2-deoxy-2-fluoro)-D-galactose and substrate activity with UDP-galactopyranose mutase

The novel UDP-sugar uridine 5'-(3-deoxy-3-fluoro-D-galactopyranosyl diphosphate) (1) and UDP-(2-deoxy-2-fluoro)-D-galactose (2) have been prepared enzymatically and tested as substrate analogues for the enzyme UDP-galactopyranose mutase (UDP-Galp mutase EC 5.4.99.9). Turnover of both 1 and 2 by UDP-Galp mutase was observed by HPLC and 19F NMR. The HPLC elution profile and 19F chemical shift of the products are consistent with the formation of the predicted furanose forms of 1 and 2. The Km values for compounds 1 and 2 were similar to those of the natural substrate UDP-Galp (0.26 mM for 1, 0.2 mM for 2, and 0.6 mM for UDP-Galp), but the values for kcat were substantially different (1.6/min for 1, 0.02/min for 2, and 1364/min for UDP-Galp). A correlation was also observed between the equilibrium yield of product formed during turnover of UDP-sugar by UDP-Galp mutase (UDP-Galp, compound 1 or compound 2), and the amount of furanose present for the free sugar at thermal equilibrium in aqueous solution, using 1H and 19F NMR spectroscopy. The implications of these results to the mechanism of the unusual enzymatic reaction are discussed.     

Separation of bisbenzylisoquinoline alkaloids by micellar electrokinetic chromatography

The micellar electrokinetic chromatographic (MEKC) separation of seven bisbenzylisoquinoline alkaloids has been developed. The effects of various separating factors were studied. Optimum separation was achieved using a buffer (pH 9.2) of 20 mM sodium borate and 20 mM sodium dihydrogen phosphate buffer containing 55 mM sodium cholate; the optimum voltage and injection time were 21 kV and 0.05 min, respectively. Highest peak efficiency was obtained when the analytes were dissolved in 10 mM sodium dodecyl sulphate as sample matrix for injection. The elution order of the bisbenzylisoquinoline alkaloids was related to their lipophilicity. The resolution, run time and detection limits of the MEKC method were compared with those of an HPLC method developed previously.     

Purine metabolism: determination of adenyl deaminase in human lymphocytes

Adenylic acid (AMP) deaminase is a "catabolic enzyme" involved in nucleotide degradation, transforming AMP into inosinic acid (IMP). We present a simple method for the determination of the enzyme activity, which combines high sensitivity with requirement of low quantities of lymphocytes. Human lymphocytes were isolated with a Lymphocyte Separation Medium from FLOW and sonicated. After centrifugation at 2,000 rpm x 10 min and treatment with Norit A, the cells were incubated at 37 degrees C with ATP 0.8 mM and 14C-AMP 0.1 mM (specific activity 12 microCi/mumole) in potassium phosphate 100 mM (pH 7.4). 14C-IMP and 14C-AMP were separated through HPLC by an isocratic elution, with 20 mM KH2PO4 (pH 5.5) at a 1.5 ml/min flow rate. Identification of the nucleotides was carried out through retention time, coelution with internal standards: their evaluation by determining the radioactivity of the collected peaks. The enzyme activity is decreased in patients affected by CLL: the decrease is evident only when data are referred to the single cells and not when they are referred to the protein.     

Separation by high-performance liquid chromatography of penicillins with C4 to C10 aliphatic side chains

A suitable and rapid method for the simultaneous determination of different aliphatic penicillins is described. Butyryl-, pentanoyl-, hexanoyl-, heptanoyl-, octanoyl-, nonanoyl- and decanoylpenicillin can be completely separated by high-performance liquid chromatography using an isocratic elution mode (50 mM H2KPO4, pH 5.0:methanol, 45:55 v/v). Under these conditions, retention times for C4 to C10 aliphatic side-chain penicillins were 2.5, 2.8, 4.1, 5.8, 8.9, 15.3, and 28.2 min. The benzylpenicillin retention time was 3.3 min.     

HPLC analysis of estrogen receptor by a multidimensional approach

Previously we demonstrated the polymorphism of estrogen receptors (ER) in cytosol of various tissues based upon properties of size, shape and surface charge. This study describes the application of a multidimensional approach utilizing HPLC for characterization of ER. Cytosols from human uterus and endometrial carcinomas were characterized sequentially by high performance size exclusion chromatography (HPSEC) on Spherogel TSK-3000 SW, and high performance ion-exchange chromatography (HPIEC) using SynChropak AX-1000 anion exchange columns. Using HPSEC, specific estrogen binding was exhibited by a 30 A isoform and by one appearing after the V0 (approximately 60 A) in human uterus. However, in endometrial carcinoma other smaller binding components with Stoke's radii of less than 20 A were observed also. In buffers containing 400 mM KCl, predominantly a 28-30 A species was observed by HPSEC. Further characterization of the 28-30 A isoform from low and high salt elution from HPSEC was accomplished with an AX-1000 column. With either condition, 2 forms were eluted on HPIEC, 1 in the column wash (retention time 8-9 min), and the other at 50-70 mM phosphate. The elution profile of the larger species (approximately 60 A by HPSEC) on the ion-exchange column was time dependent. Immediate analysis (within 15 min) showed a profile similar to that of the original cytosol which contained minor components eluting in wash buffer and at 50-70 mM phosphate and a major isoform at 180 mM phosphate. However delayed analysis (after 2 h) of the 60 A isoform showed a similar profile (components in buffer wash and at 50-70 mM phosphate) obtained with the 30 A species. This time dependent change was not observed for the 30 A species or for the original cytosol. Estrogen receptors in cytosol sedimented at 10S and 4S in low ionic strength gradients and at 4S in sucrose gradients containing 400 mM KCl. The 28-30 A and 60 A species recovered from HPSEC sedimented at 3.5S. This multidimensional approach indicates that native estrogen receptors dissociated into a number of smaller molecular isoforms, which were distinguishable by different surface charge properties.     

Putative androgen receptors distinguished in wild-type and testicular-feminized (Tfm) mice.

The reduced level of putative androgen receptor in the mouse mutant, testicular feminization (Tfm), chromatographs on DNA-cellulose differently from the bulk of wild-type receptors. While the elution maximum for extracts of Tfm/Y kidney is in the 180–190 mM NaCl range, wild-type kidney extracts exhibit two maxima of elution at 140–150 mM NaCl and 180–190 mM NaCl, respectively. For hypothalamus-preoptic area, Tfm/Y has one elution maximum at approximately 180 mM NaCl, while the wild-type exhibits a major elution maximum at 140–150 mM NaCl, with a minor peak at approximately 180 mM NaCl. Mixing experiments between wild-type and Tfm/Y cytosols reveal that the different characteristic elution patterns are intrinsic to the binding complexes and are not conveyed simply by other soluble factors. The distinctive pattern for Tfm indicates that the mutation does not cause merely a reduced level of wild-type receptor. Rather the residual receptor of the mutant may be either an abnormal protein or a minor form of wild-type receptor, not readily seen in wild-type tissue due to the presence of more preponderant species. Differences in the elution profiles of androgen receptor species of wild-type kidney with the two bound androgens, testosterone and dihydrotestosterone, are also presented. A model of the androgen receptor system is proposed which includes several binding classes for androgen ligands and metabolites. In light of aromatization of androgens to estrogens and its probable role in some androgenic responses, we include the “estrogen receptor” in this mechanism.     

Fast and sensitive determination of urinary 1-hydroxypyrene by packed capillary column switching liquid chromatography coupled to micro-electrospray time-of-flight mass spectrometry

The present work reports capillary liquid chromatographic column switching methodology tailored for fast, sensitive and selective determination of 1-hydroxypyrene (1-OHP) in human urine using micro-electrospray ionization time-of-flight mass spectrometric detection. Samples (100 microl) of deconjugated, water diluted and filtered urine samples were loaded onto a 150 microm I.D.x 30 mm 10 microm Kromasil C(18) pre-column, providing on-line sample clean-up and analyte enrichment, prior to back flushed elution onto a 150 microm I.D.x 100 mm 3.5 microm Kromasil C(18) analytical column. Loading flow rates up to 100 microl/min in addition to the use of isocratic elution by a mobile phase composition of acetonitrile/water (70/30, v/v) containing 5 mM ammonium acetate provided elution of 1-OHP within 5.5 min and a total analysis time of less than 15 min with manual operation. Ionization was performed in the negative mode and 1-OHP was observed as [M-H](-) at m/z 217.08. The method was validated over the concentration range 0.2-40 ng/ml 1-OHP in pre-treated urine, yielding a coefficient of correlation of 0.997. The within-assay (n=6) and between-assay (n=6) precisions were in the range 6.4-7.3 and 7.0-8.1%, respectively, and the recoveries were in the range 96.2-97.5 within the investigated concentration range. The method mass limit of detection was 2 pg, corresponding to a 1-OHP concentration limit of detection of 20 pg/ml (0.09 nmol/l) diluted urine or 0.3 ng/ml (1.35 nmol/l) urine.     

Determination of sulphasalazine and its main metabolite sulphapyridine and 5-aminosalicylic acid in human plasma by liquid chromatography/tandem mass spectrometry and its application to a pharmacokinetic study

A simple and sensitive liquid chromatography/positive-ion electrospray ionization mass spectrometry (LC-ESI-MS/MS) method has been developed for the simultaneous determination of sulphasalazine (SASP) and its main metabolite sulphapyridine (SP) and 5-aminosalicylic acid (5-ASA) with 100 μL of human plasma using dimenhydrinate as the internal standard (I.S.). The API-3000 LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Protein precipitation process was used to extract SASP, SP, 5-ASA and I.S. from human plasma. The total run time was 9.0 min and the elution of SASP, SP and 5-ASA was at 4.8 min, 2.5 min and 2.0 min, respectively. The separation was achieved with a mobile phase consisting of 0.2% formic acid, 2 mM ammonium acetate in water (mobile phase A) and 0.2% formic acid, 2 mM ammonium acetate in methanol (mobile phase B) by using gradient elution on a XBP Phenyl column (100 mm × 2.1 mm, 5 μm). The developed method was validated in human plasma with a lower limit of quantitation of 10 ng/mL for SASP, SP and 5-ASA, respectively. A linear response function was established for the range of concentrations 10-10,000 ng/mL (r>0.99) for SASP and 10-1000 ng/mL (r>0.99) for SP and 5-ASA. The intra and inter-day precision values for SASP, SP and 5-ASA met the acceptance as per FDA guidelines. SASP, SP and 5-ASA were stable during stability studies, i.e., long term, auto-sampler and freeze/thaw cycles. The method was successfully applied for the evaluation of pharmacokinetics of SASP, SP and 5-ASA after single oral doses of 250 mg SASP to 10 healthy volunteers.     

Separation and quantification of muropeptides with high-performance liquid chromatography

About 80 different muropeptides, the subunits which comprise the polymer murein of Escherichia coli, were resolved by high-performance liquid chromatography. The muropeptides were released from isolated murein by complete digestion with muramidase from Chalaropsis spec. The separation method is based on reversed phase chromatography of the sodium borohydride-reduced compounds using ODS (C18) columns and a linear gradient elution with sodium phosphate buffer and methanol as organic modifier. The effect of temperature, pH, ionic strength, and the steepness of the gradient and of different support materials on the separation of the muropeptides was investigated. The new method represents a major improvement over previous methods with respect to resolution, sensitivity, and speed. Analytical as well as preparative separations can be realized. Quantitative analysis of murein composition is achieved by a linear gradient from 50 mM sodium phosphate, pH 4.31, to 75 mM sodium phosphate, pH 4.95, containing 15% methanol for 135 min on a 250 X 4.6 mm 3-micron Hypersil ODS column at 55 degrees C using a flow rate of 0.5 ml/min. With uv detection at 205 nm about 20 micrograms of murein per analysis is sufficient. The detection limit per compound is about 5 ng. A method for the evaluation of the analytical data allowing a convenient comparison of different muropeptide pattern is described.     

Development and validation of a new reversed-phase ion pairing liquid chromatographic method with fluorescence detection for penciclovir analysis in plasma and aqueous humor

A simple, sensitive and reliable HPLC ion-pairing method with fluorescence detection, was developed for penciclovir determination in plasma and aqueous humor, with a Zorbax SB-aq C18 (100 mmx2.1 mm) column. Plasma samples were treated by solid-phase extraction with Oasis MCX (30 mg) cartridges. Ganciclovir, an antiviral drug structurally related to penciclovir, was used as internal standard (I.S.). Aqueous humor samples were directly injected into the chromatographic system. Separation was performed by a gradient elution with a mobile phase consisting of a mixture of acetonitrile and phosphate buffer 50mM containing 5mM of sodium octanesulfonate, pH 2.0, at a flow rate of 0.3 ml/min. The method was validated and showed good performances in terms of linearity, sensitivity, precision and trueness. Quantification limit was obtained at 0.05 microg/ml for aqueous humor and at 0.1 microg/ml for plasma. Finally, the proposed analytical method was used to measure penciclovir in clinical samples for a pharmacokinetic study, after oral administration of famciclovir.     

Integration of bacteria capture via filtration and in situ lysis for recovery of plasmid DNA under industry-compatible conditions

Combining capture and lysis of the bacteria with partial purification of the plasmid DNA is beneficial for the design of efficient plasmid production processes at larger scale. Such an approach is possible when the bacteria are captured by filtration. Taking industrial requirements into account, however, such a capture requires complex filtration mixtures containing retentive additives such as bentonite and polycations. This makes the straightforward transfer of established lysis protocols to in situ lysis difficult. In this contribution, the different steps of such a protocol are designed for complex filter cakes, including fragilization (by lysozyme), lysis (alkaline pH/acidic pH, 70/37 degrees C, urea/NaCl/Triton), and specific elution (pH, NaCl, CaCl2, guanidinium hydrochloride). Results are compared in regard to plasmid quality (topoisomeric form) and quantity (compared to the yield obtained by a commercial miniprep of a small aliquot of the bacteria suspension from the bioreactor). Best results in these terms were obtained by the Triton lysis protocol performed at 37 degrees C (30 min of contact with a lysis buffer composed of 50 mM Tris pH 8, 1% Triton, 1 g/L lysozyme, and 6 M guanidinium hydrochloride) followed by the specific elution of the plasmid DNA in 50 mM Tris buffer pH 8.     

YlyA和RNA聚合酶的反应性鉴定

目的：用亲和层析法鉴定YlyA与RNA聚合酶（RNAP）的结合性能。方法：将YlyA分别上样于以Affigel 15为亲和介质制备的空白柱、牛血清白蛋白（BSA）柱和RNAP柱;以GreA和绿色荧光蛋白（GFP）为阳性和阴性对照蛋白分别上样于同一RNAP柱,洗涤和洗脱缓冲液（pH均为7．9）的盐离子浓度分别为30mmol／L和400mmol／L;用免疫印迹法对洗涤和洗脱流出液中的YlyA进行检测。结果：在空白柱和BSA柱的洗脱收集液中,没有检测到YlyA,大量的YlyA出现在了洗涤收集液中;而在RNAP柱的洗脱收集液中,检测到了YlyA和GreA,没有检测到GFP。结论：YlyA与RNAP之间具有特异性结合能力,为YlyA极有可能是一种转录因子的生物信息学分析结果提供了实验证据。     

Purification and properties of cytoplasmic and mitochondrial malate dehydrogenases of Physarum polycephalum

Two isoenzymes of malate dehydrogenase (MDH) were demonstrated in plasmodia of Physarum polycephalum by polyacrylamide-gel electrophoresis. The more "cathodal" form was uniquely associated with mitochondria (M-MDH) and the other form was found in the soluble cytoplasm (S-MDH). The isoenzymes were separated by acetone fractionation of soluble plasmodial homogenates acidified to pH 5.0. The M-MDH was purified 201-fold by cetylpyridinium chloride treatment, fractionation with ammonium sulfate, gradient elution from sulfoethyl cellulose at pH 6.0, and Sephadex G-100 chromatography. The S-MDH was purified 155-fold by ammonium sulfate fractionation, diethylaminoethyl cellulose chromatography, gradient elution from sulfoethyl cellulose at pH 5.5, and Sephadex G-100 chromatography. The optimal cis-oxalacetate concentrations were 0.35 mM for M-MDH and 0.25 mM for S-MDH, and the optimal pH for both isoenzymes was 7.6 for oxalacetate reduction. The optimal l-malate concentrations were 5 mM for S-MDH and 6 mM for M-MDH, and both isoenzymes exhibited an optimal pH of 10.0 for L-malate oxidation. The Michaelis constants of S-MDH and M-MDH served to discriminate between the isoenzymes. The S-MDH was more heat-stable than the M-MDH. High concentrations of oxalacetate and malate inhibited S-MDH more than M-MDH. The isoenzymes were further distinguished by their utilization of analogues of nicotinamide adenine dinucleotide. Many properties of the Physarum isoenzymes were similar to those of more complex organisms, especially vertebrates.     

Chromatographic behavior of Bothrops erythromelas phospholipase and other venom constituents on Superdex 75

In a chromatographic method modification intended to preserve protease activity in Bothrops erythromelas venom, 2 mM CaCl2 was added to the gel filtration buffer [50mM Tris/HCl/150mM NaCl (pH 8.0)], in lieu of an equimolar portion of NaCl. This minor compositional change induced significant differences in the venom elution profile on Superdex 200. For this reason, the influence of buffer composition on chromatographic behavior was investigated using an analytical Superdex 75 HR 10/30 column. Phospholipase (PLA) was used as a marker because Naja atra PLA had previously been observed to interact hydrophobically with this resin. PLA elution volumes generally increased as buffer pH decreased. Addition of 20% acetonitrile to the Tris buffer with CaCl2, reduced hydrophobic interaction of the PLA so significantly that its elution was non-overlapping in the two buffers. Other venom constituents, including bradykinin-potentiating peptides and probable hemorrhagic metalloproteases, were similarly affected. Buffer calcium, bound by vicinal dextran hydroxyl groups, appears to retard elution of this acidic PLA.     

Relationship between MPA free fraction and free MPAG concentrations in heart transplant recipients based on simultaneous HPLC quantification of the target compounds in human plasma

A simple and sensitive HPLC method for the simultaneous analysis of free MPA and free MPAG was developed. Separation was achieved on a X-Terra RP18 column with acetonitrile-40 mM orthophosphoric acid as eluents using a gradient elution mode over 35 min at a flow rate of 1.5 ml/min. The assay was linear in the range 0.005 mg/L (LOQ) to 5mg/L for free MPA and 0.05 mg/L (LOQ) to 200 mg/L for free MPAG. Isolation of free MPA and free MPAG was done by ultrafiltration and the ultrafiltrate was directly injected. A positive correlation between MPA free fractions and free MPAG concentrations was found. Likewise, free MPAG was related to total MPAG concentrations in the seven heart transplant patients.