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IntroductionDissolution is the amount of substance that goes into solution per unit time under standardised conditions of liquid/solid interface, solvent composition and temperature. Dissolution is one of the most important tools to predict the in-vivo bioavailability and in some cases to determine bioequivalence and assure interchangeability.AimTo compare the differences in dissolution behaviour of solid dosage forms between innovators (reference products) and their generic counterparts (tested products).MethodsFour replicates for each batch of 37 tested medicines was carried out using A PT-DT70 dissolution tester from Pharma Test. A total of 13 branded medicines and 24 generic counterparts were obtained locally and internationally to detect any differences in their dissolution behaviour. They were tested according to the British Pharmacopeia, European Pharmacopeia and the US Pharmacopeia with the rate of dissolution determined by ultra-violet Spectrophotometery.ResultsMost tested medicines complied with the pharmacopoeial specifications and achieved 85% dissolution in 60min. However, some generic medicines showed significant differences in dissolution rate at 60 and 120min. Many generic medicines showed a slower dissolution rate than their branded counterparts such as the generic forms of omeprazole 20mg. Some showed an incomplete dissolution such as the generic form of nifedipine 10mg. Other generics showed faster dissolution rate than their branded counterpart such as the generic forms of meloxicam 15mg. Moreover, some generics from different batches of the same manufacturer showed significant differences in their dissolution rate such as the generic forms of meloxicam 7.5mg. Nevertheless, some generic medicines violated the EMA and the FDA guidelines for industry when they failed to achieve 85% dissolution at 60min, such as the generic form of diclofenac sodium 50mg.ConclusionMost medicines in this study complied with the pharmacopeial limits. However, some generics dissolved differently than their branded counterparts. This can clearly question the interchangeability between the branded and its generic counterpart or even among generics.

This study investigated the biochemical relationship between human follicular/oocyte maturity and the levels of follicular fluid purines. Intrafollicular levels of purine metabolites and creatinine are associated with oocyte presence, and the presence of such high levels of adenosine indicates a privileged site with no adenosine deaminase activity. Subgrouping according to oocyte recovery and fertilization revealed differences in correlation between the purine metabolites: Only where an oocyte was recovered and subsequently fertilized did follicular fluid adenosine, adenine, and hypoxanthine levels correlate with each other. Significantly, purines' correlation with levels of the terminal degradation product, uric acid, could only be seen in failed fertilization samples. Given the established metabolic pathways for adenosine triphosphate/adenosine diphosphate/adenosine monophosphate degradation, the results indicate maximization of 2 purine salvage pathways (from adenine and hypoxanthine) that pivot on the presence of high adenosine levels. Such optimized recovery may be necessary to build a store of salvaged adenosine phosphate for oocyte survival.

Some of the major serum proteins that are also found in follicular fluid, including transferrin, alpha-macroglobulin and albumin, are thought to play a role in oocyte maturation. This study set out to identify proteins in human follicular fluid by capillary zone electrophoresis and to investigate their relationship to follicular/oocyte maturity and fertility outcome. 176 individual follicular fluid samples, from 30 women undertaking in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI), were run using an optimized capillary zone electrophoresis method that gave a good separation of sixteen peaks in most samples. Nine of the peaks were identified and quantified but seven remain unknown and require further proteomic identification. Of the identified protein peaks, levels of each were corrected for follicular volume and total content calculated. No significant difference in protein levels was found with regard to oocyte recovery and fertilization. Protein concentrations tended to decrease as the follicular sphere increased whilst total content in follicular fluid increased in proportion to size. This is consistent with simple transudation across a sphere surface area which does not increase in proportion to the follicular fluid. This is not true of the concentration and content pattern of other proteins/biomolecules which are produced by follicular cells locally. In conclusion, neither concentration nor absolute levels of nine major proteins identified in follicular fluids correlated with oocyte presence and fertility outcome. Future work to remove more concentrated proteins (e.g. albumin) would enhance separation of smaller peaks and identification of the unknown molecules.

Study Objective To characterize the insoluble particulate matter generated during hysteroscopic monopolar and bipolar electrosurgery. Design In vitro comparative study (Canadian Task Force Classification II-1). Setting Teaching hospital. Patients Eight patients undergoing hysterectomy with benign indications. Intervention Tissue blocks were divided, and 1 g of tissue was vaporized using a monopolar and a bipolar vaporizing electrode. Measurements and Main Results The particles were examined at light microscopy to study their morphologic features. Particle analysis was carried out using a laser diffraction particle analyser. Monopolar uterine tissue vaporization generated particles of a smaller median diameter than did bipolar vaporization (0.13 μm and 0.25 μm, respectively). The distribution by particle surface area in both instruments seems to be complex, with peaks at 0.35 and 4.0 μm for monopolar-generated particles and 0.5 and 5.0 μm for bipolar-generated particles. Another smaller peak with larger particles produced by both instruments was identified at 30 μm. During monopolar uterine tissue vaporization, 9.96% of particles generated were smaller than 5 μm, whereas during bipolar vaporization, 3.81% of particles were smaller than 5 μm (p = .01). Conclusion Particulate matter generated during hysteroscopic electrosurgical vaporization using monopolar and bipolar instruments is heterodisperse and complex in shape. Monopolar vaporization results in particles of a smaller median diameter compared with bipolar vaporization. Both instruments generate 2 distinct populations of particles by surface area. The distribution of particles by volume during hysteroscopic electrosurgery is complex.

In our experience the efficient design of molecularly imprinted polymer (MIPs) for novel templates has proved difficult. Following commonly used imprinting protocols, MIPs designed against one template show both a lack of capacity and poor specificity for rebinding either the template or structurally similar analytes. Optimisation methods that involve changing one factor at a time can be laborious. A novel approach for the optimisation of MIPs using chemometrics is described. Sulfonamides, common drug residues in foodstuffs, were used as the model analytes with a methacrylic acid/ethylene glycol dimethacrylate MIP. To avoid the inaccuracies in measurement caused by template bleed a multi-analyte competition rebind assay was developed to select suitable sulfonamides to be used as the template for the MIP, and for the rebind analyte in the chemometric optimisation study. The rebinding efficiencies were monitored by HPLC. The template sulfonamide was selected as sulfamethazine (SMZ), and the rebind analyte as sulfadimethoxine (SDIM). The template:monomer:cross-linker (T:M:X) ratio of the SMZ block MIP was then optimised using a three-level full factorial design to predict a MIP with the highest rebind capacity. On synthesis this was 38.8% for SDIM in a solid phase extraction (SPE) application agreeing with the predication. The factorial design was further utilised to predict an optimum T:M:X ratio for the production of a class specific MIP, capable of binding a range of sulfonamides simultaneously. The predicted optimum T:M:X ratios of (1:10:55) and (1:10:10) were found to be different to commonly used ratios from the MIP literature.

A novel triple column capillary electrophoresis system is described. Design specifications facilitate method development and analyses by providing on-line, selective, pre-concentration and clean-up of both high (ml) and low (μl) volumes of specific analytes in two dimensions and separation via an additional third dimension. The system described additionally provides four distinct detection capabilities via both contactless conductivity and UV. The addition of a third dimension to the previously reported “coupled-column” systems, and further modifications made, has allowed for optimal identification, separation, and quantitation of micro-components in complex mixtures. The ability to perform both capillary zone electrophoretic and isotachophoretic separations on-line and in any combination enhances the scope for rapid analytical method development and analysis of complex or trace sample components.

Experimental colitis is associated with anorexia that is attenuated by treatment with an interleukin (IL)-1 receptor antagonist. Serotonin (5-hydroxytryptamine [5-HT]) is a potent inhibitor of feeding, and its release from the hypothalamus is stimulated by IL-1. We have tested the hypotheses that anorexia associated with experimental colitis results from increased activity of hypothalamic 5-HT neurons and that the increase in activity occurs secondary to an increase in availability of tryptophan, the precursor of 5-HT.In vivo 5-HT release and regional hypothalamic 5-HT and tryptophan concentrations were measured in rats with 2,4,6,-trinitrobenzene sulfonic acid (TNBS)-induced colitis, healthy controls, and animals pair-fed to match the food intake of the colitic group. Food intake in the colitic group was assessed after depletion of brain 5-HT by p-chlorophenylalanine (PCPA).In the colitic group, release of 5-HT from the hypothalamic paraventricular nucleus (PVN) was 3-fold (P = 0.01) and 14-fold (P0.001) higher than in control and pair-fed groups, respectively. Concentrations of tryptophan were similar in each group in all hypothalamic regions. Food intake was significantly increased in the colitic group after PCPA treatment but was not restored to control values.In animals with TNBS-induced colitis, 5-HT release from the PVN is increased. The increase in food intake after depletion of brain 5-HT suggests that hypothalamic 5-HT contributes to anorexia but is not the only mediator. Increased 5-HT release in the colitic group was not driven by increased precursor availability.

A micellar electrokinetic chromatographic method is described for the determination and quantitation of allantoin, an end-product of purine metabolism in mammals that is applicable to biofluids of different mammal species and man. The method was optimised following a study on the effect of pH and sample preparation procedure. Final conditions were 30 mM sodium tetraborate, pH 9.5, 75 mM sodium dodecyl sulphate, 20 kV and 20 degrees C. Allantoin was well resolved from endogenous compounds and could be determined in horse, dog, mouse and rabbit urine. No allantoin could be found in man. No complicated sample treatment was necessary, thus the developed method was rapid (5 min), sensitive (5 microM) and simple. Results from this work will permit the determination of allantoin in man as a measure of free radical generation reactions as well as its presence in the plasma and other biofluids with modification of the sample preparation procedures.

There are many high-performance liquid chromatographic assays for the determination of sulphonamides, dihydrofolate reductase inhibitors and β-lactam antibiotics individually in food products. Recently, some capillary electrophoresis (CE) assays for these drugs have appeared. Sulphonamides plus dihydrofolate reductase inhibitors are readily separated by capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) can separate β-lactams. However, if CE is to prove its value over HPLC techniques in practice then its intrinsic high resolution must be exploited fully. A combination of a sulphonamide-dihydrofolate reductase inhibitors separation by CZE with a MECC assay of β-lactams would be advantageous in the screening of food products for veterinary drug residues, saving both time and effort. The analysis of all three classes of drugs, in a single run, was tested using our established β-lactam MECC conditions, but this gave only a partial separation, with a very crowded region. A Box-Behnken factorial design was used to optimise this separation. It was carried out parallel with the “traditional trial and error procedure”, based on our knowledge of the individual CE separations. Both methods arrived at a remarkably similar set of conditions. The final separation produced 25 peaks, within which, were 30 compounds, the separation taking 25 min.

This paper demonstrates the use of micellar electrokinetic capillary chromatography (MECC) to monitor enzyme reaction conditions. The hydrolysis reactions of model conjugated substrates (morphine and reduced flunixin glucuronides, napthyl sulfate), by proprietary beta-glucuronidase preparations, were studied under varied experimental conditions. Reactions were carried out in autosampler vials with incubation in a thermostatted CE autosampler tray. MECC was performed using borax buffer (17.5 mM, pH 9.3) modified with sodium dodecyl sulfate (70 mM). Repetitive injections were made from the sample vial throughout the course of the reactions at a frequency of up to 10 h-1. MECC provided a rapid and reproducible assay for the model substrates. Baseline interference from the enzymes prevented measurement of product increase, therefore substrate decrease was measured from the peak areas. Monitoring of reactions in this way has proved valuable in the optimisation of hydrolysis conditions used in sample preparation for drug analysis. beta-Glucuronidase preparations from Helix pomatia were found to give the best performance of those evaluated in terms of deconjugation efficiency.

Micellar electrokinetic capillary chromatography (MECC) using diode array detection has been investigated for the determination of phase I and phase II metabolites of drugs in biofluids. Methods were optimised for the determination of morphine, morphine-3-glucuronide, morphine-6-glucuronide, normorphine, meclofenamic acid and its metabolites in equine urine. Solid-phase extraction procedure were developed to concentrate and purify the analytes from spiked and post administration urines for MECC analysis. A simple on-line procedure for monitoring the kinetics of hydrolysis of morphine-glucuronide conjugates by beta-glucuronidase was demonstrated.

Purine nucleotide metabolism has been studied in T-lymphocyte population in healthy subjects and in AIDS bearing patients. Nucleotide content was determined by HPLC. The overall rate of purine nucleotide synthesis was measured following the incorporation of 14C-formate into the nucleotides of a T cell suspension. The authors discuss the results, which indicate interesting variations in nucleotide content and a lower nucleotide synthesis, determined by kinetic studies.

Characterization of haemoglobin (Hb) through whole protein separations and tryptic digest mapping allows the identification of structural Hb variants which result in haemoglobinopathies. Tryptic digest mapping by conventional two-dimensional paper chromatograph-electrophoresis provides high resolution but requires 48 h, while gradient elution reversed-phase high-performance liquid chromatography (HPLC) is faster (1.5 h), there is decreased resolution. CE analysis provides a fast separation with high resolution. We have used CE to optimise the tryptic digestion of globin purified from normal human haemoglobin A and to analyze tryptic digests from normal Hb. The separations were optimised and peak identification performed using UV scanning detection. In the optimised tryptic digest separation up to 28 peaks could be resolved in < 20 min. These peaks were identified as far as possible and a high-resolution map of the digest was constructed. The optimised analytical conditions were used to observe the separation pattern obtained from normal adult haemoglobin (HbA), common variant haemoglobins and some rarer haemoglobin variants.

An optimized separation of the main purine compounds of human serum by capillary zone electrophoresis is presented. Separations were performed in an uncoated silica capillary (44 cm x 75 microns I.D., 37 cm to window) on a SpectraPhoresis 1000 system with UV detection. The separation of adenine (Ade), adenosine (Ado), guanine (Gua), guanosine (Guo), hypoxanthine (Hyp), inosine (Ino), xanthine (Xan) and uric acid (UA) was optimized with respect to pH, temperature, applied potential and hydrodynamic injection time. Optimum conditions were 20 mM borate buffer (pH 9.4), 37 degrees C, 20 kV and 9 s load and detection at 260 nm. Linearity extended from 1 to 125 microM. The sensitivity of the method was 0.5 microM, which is adequate for measuring Ade, Gua, Hyp and UA in plasma samples. Plasma samples from newborns were precipitated with an equal volume of perchloric acid (7%, v/v), the supernatant was adjusted to neutral pH with potassium carbonate and, before injection, the sample was alkalized with sodium hydroxide. The method presented here allows the determination of Ade, Guo, Hyp and UA. The levels of the determined purines were compared in samples from control newborns, preterm babies and newborns with asphyxia or acidic serum pH values.

The pyridinium crosslinks are important biomarkers of mature hard tissue collagen degradation. This paper describes an isocratic ion-paired reversed-phase high-performance chromatographic assay using narrow-bore columns and high-sensitivity fluorescence detection to enable for the first time the determination of pyridinium crosslinks in both serum and synovial fluid samples. Extracted freeze-dried acid hydrolysates were re-suspended in 20 mM pentafluoropropionic acid (PFPA). Separations were carried out using an Exsil 100 5-μm ODS2 column (100 mm × 2.1 mm I.D.) eluted with 10 mM PFPA in water at 0.15 ml/min and detected using a Jasco 821-FP detector (xenon lamp: excitation 290 nm, emission 400 nm). Fluorescent response was linear from 269 to 8620 fmol for pyridinoline (Pyr) and 85 to 2710 fmol for deoxypyridinoline (dPyr). The limits of detection were 28 and 57 fmol, respectively. The coefficient of variation for extraction and analysis of normal serum was 7.96% for Pyr and 6.30% for dPyr (n = 6). The mean ± S.D. concentration of Pyr in normal serum was 3.26 ± 0.83 nM.

Capillary electrophoresis (CE) is now at a critical stage in its development when, as with all new analytical techniques, it needs to be accepted by a wider research community and be usefully employed in other disciplines including biomedicine. It offers many attractions to the bioanalyst seeking to separate charged molecules; however, many questions remain unanswered with regard to its applicability and usefulness. In this critical overview we combine literature data with some of our own observations with regard to the application of CE to the separation, detection and quantitation of bio-molecules and compare them to our overall and much longer experience with high-performance liquid chromatography. Finally we speculate on the improvements and direction we might like CE to take with respect to the analysis of molecules in biofluids.

A simple nucleotide analyser was constructed from commercially available components. A 20 cm × 0.4 cm I.D. column of pellicular anion exchanger operating at approximately 20 p.s.i. was found to perform identically with published methods using high-pressure equipment. The eluant was monitored using a variable-wavelength UV monitor. A very simple device for producing variable gradients was employed. The nueclotide analyser was trouble free to operate and the results were both linear and reproducible. Applications to the rapid analysis of nucleotides and the high-sensitivity analysis (approx. 10 pmoles) of cyclic 3′,5′-adenosine monophosphate and cyclic 2′,3′-guanosine monophosphate and tissue nucleotides are described.

A rapid and sensitive assay for serum cytidine deaminase has been developed utilising ion-pair reversed-phase high-performance liquid chromatography. The addition of 1-octanesulphonic acid (OSA) caused the retention of cytidine and uridine to reverse and uridine, the minor component in the assay, to elute first. Cytidine, uridine and allopurinol (internal standard) were separated on a 5-micron Hypersil ODS column using 100 mM ammonium acetate with 1% (v/v) methanol and 1 mM OSA adjusted to pH 5.0. Detection was at 262 nm. Peak areas were linear from 7 pmol to 6 nmol injected (r = 0.99). Intra-assay variation was 7.8% (n = 10) and the correlation with a colorimetric assay was r = 0.78 (p less than 0.001).

The ATP ADP ratio, measured by high performance liquid chromatography, has been used as an indicator of stability of erythrocyte nucleotides. The nucleotides from human, rabbit and rat whole blood, but not separated erythrocytes were stable for maximum periods of 40, 20 and 15 min respectively after venepuncture. The ratios then declined rapidly from 9 to 5, 12 to 4 and 9 to 1 respectively during 2h storage at room temperature. Similar changes occurred in GTP GDP ratios. The relevance of these observations to metabolic studies in intact cells, nucleotide analyses in the clinical situation and comparative studies in other species is discussed.

A case is reported of a patient with idiopathic hypercalcuria who was referred for investigation of renal calculi. Studies of urinary amino acid excretion in the patient and all living members of his family, as well as studies of endogenous renal clearances of dibasic amino acids and cystine in the patient and his daughter, indicated that the patient had genotype +/11 heterozygous cystinuria. Intestinal perfusion studies showed however that the patient was able to absorb L-lysine, L-arginine normally from the jejunum and ileum. This suggests that a difference exists between renal and intestinal handling of lysine and arginine in cystinuria.

The differing effect of pH on the reaction of fluorescamine with amino acids and peptides has been utilized to develop a method for the quantitative estimation of peptides in the presence of amino acids. This method has been applied to the measurement of dipeptidase activities in human jejunal aspirates.

A method is described for the preparation of concentrated tissue extracts for nucleotideanalysis by high-performance liquid chromatography (hplc). Ten to one hundred milligrams of tissue was extracted in a combined weighing-homogenizing-centrifuge tube using a trichloracetic acid (TCA)-methanol extractant containing a radioactive internal standard. This extractant eliminated nucleotide interconversion which was found to occur when TCA alone was employed. High ATP/ADP and ATP/AMP ratios were observed and recoveries of greater than 97% were obtained with exogenous radioactive nucleotides. The method has been applied successfully in studies on muscle, heart, liver, kidney, lung, brain, and subcellular fractions.

An unknown metabolite of D -penicillamine found in the urine of patients receiving the drug has been isolated by ion-exchange chromatography. It was identified as S-methyl- D -penicillamine by mass spectrometry and its structure was confirmed by synthesis. In subjects receiving D -penicillamine for treatment of cystinuria and rheumatoid arthritis 4 and 8 per cent respectively of the dose was methylated. The total percentage of the D -penicillamine dose excreted (as cysteine-penicillamine plus penicillamine disulphide plus S-methyl- D -penicillamine) was 40 and 34 per cent in cystinuria and rheumatoid arthritis respectively. The findings in two cases of Wilson's Disease were similar to those in rheumatoid arthritis. In studies on four cystinuria patients an average of 12 per cent of the administered D -penicillamine was recovered in the faeces mainly as penicillamine disulphide. However, only between 42 and 53 per cent of the dose was identified in the urine and faeces, so that approximately 50 per cent of the administered drug was unaccounted for. D -Penicillamine caused a 32 per cent reduction in the urinary excretion of cysteine residues in cystinuria but a 400 per cent increase in their excretion in rheumatoid arthritis.

Ileal transport of dibasic amino acids has not previously been studied in the intestine of healthy volunteers or cystinuric patients. Experiments have therefore been designed to compare ileal and jejunal absorption of lysine and arginine both in normal subjects and cystinuric patients. In addition, jejunal perfusion experiments have been carried out to investigate absorption of the dipeptide L-arginyl-L-leucine. The results indicate that, at the concentrations studied (4.2 mm lysine, 1 mm arginine), severe transport defects exist throughout the whole small intestine for both amino acids in cystinuria. Despite the transport defect for free arginine, cystinuric patients absorbed the dipeptide l-arginyl-l-leucine normally. Because of the transport defect for free arginine, it has been possible to show that during absorption of l-arginyl-l-leucine in cystinuria approximately 30% of dipeptide-bound arginine can be recovered from the gut lumen in the free form. These findings indicate that the prime function of specific amino acid transport systems during the absorption of protein digestion products may be as a "recapture mechanism" for amino acids liberated as a result of mucosal cell peptide hydrolysis.

Adenine and adenosine metabolism has been studied in intact human erythrocytes in vitro using high performance liquid chromatography, isotopic labelling and electrophoresis. Their metabolism to nucleotides was controlled by phosphoribose diphosphate synthesis which was phosphate dependent. Adenosine formed hypoxanthine or IMP depending upon P i concentration, but adenosine kinase and deaminase activities were not affected by P i levels. Free [ 14 C]adenine and [ 14 C]hypoxanthine were found in cellular extracts. Rapid interconversions occurred to give a distribution for ATP : ADP : AMP of 10 : 1 : 0.1. Marked decomposition of ATP to ADP and AMP occurred during incubations in plasma and Earle's media in air on nitrogen, but ATP levels remained stable in phosphate buffers and in the presence of oxygen. At physiological P i (1 mM) adenosine kinase activity grossly exceeded adenine phosphoribosyltransferase activity. The latter was approximately 7 fold that of hypoxanthine phosphoribosyltransferase activity. These differences decreased with increasing P i levels. No significant increase in corresponding nucleotides was obtained by incubation with high levels (0.5 mM) of adenine, guanine or guanosine at physiological P i , ATP increased by 10% independently of the substrate employed and significant amounts of IMP and GTP were formed from adenosine and guanosine, respectively. The existence of a bound intracellular pool of ATP is suggested.

Summary The metabolism of adenosine and adenine by the intact human erythrocyte has been studied over the range 0.1 to 180 μmol/l. At physiological levels ( 90% of the adenosine was deaminated. Over the physiological range adenine and adenosine were incorporated into nucleotides at identical rates. The relevance of these findings to human purine metabolism and their implications to adenosine deaminase deficient patients is discussed.

1. 1. Langendorff perfused rat hearts show synchronous, statistically significant, systematic variations in ATP and ADP. 2. 2. Here we show that AMP and IMP also vary in register with ATP and ADP and we suggest that the synchronizing trigger for these oscillations may be ischaemia. 3. 3. Oscillations in the ATP/ADP ratio were found to be significantly correlated with creatine phosphate content but by contrast these quantities vary quite differently from the GTP/GDP ratio. 4. 4. Cyclic GMP oscillations showed a significant negative correlation with variations in ADP. 5. 5. Epinephrine raised mean cyclic AMP content and stabilized cyclic GMP oscillations, but had little other effect on the purine nucleotide variations.

A modified double lumen perfusion method has been used to study dibasic amino acid absorption in human jejunum. Results using this method have been compared with those from oral load studies. Using the perfusion method, kinetic constants (Kt and Vmax) for lysine were: Kt 4.9 mM; V max 51.3 μmoles min-1 30 cm and for arginine were: Kt 3.8 mM, V max 46.3 μmoles min-1 30 cm Lysine absorption was compared in normal and cystinuric subjects. A significant difference was seen between these groups when studied by perfusion but not using the oral load method. Furthermore, oral load studies using mixtures of lysine, ornithine, and arginine showed a difference between the groups for arginine but no difference for lysine and ornithine. All three dibasic amino acids appeared to be well absorbed in the cystinuric patients. Possible explanations for these differences and the failure of oral tolerance tests to demonstrate the known transport defect in cystinuria are discussed.