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The use of high-performance liquid chromatography (HPLC) in the control of rDNA-derived human insulin and human growth hormone is described. Powerful identity tests based upon reversed-phase HPLC separation of enzymatic digests have been developed. Size exclusion and reversed-phase assays are used to control higher molecular weight materials and monomeric derivatives, respectively, for both proteins. Finally, HPLC is used to control the relevant protein content, which in concert with other information controls the biopotency of the protein preparations.

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The applicability of amino acid analysis for accurate quantitation of reference standard preparations of proteins has been evaluated. This approach is very useful since, in addition to absolute quantitative information, it also provides a measure of composition, partial identity, and purity in a single experiment. Comparisons with Kjeldahl nitrogen assay and/or UV measurements shows that amino acid analysis is reliable for the quantitation of small-to-medium size proteins in the molecular weight range of 6-22 kDa. For larger proteins such as immunoglobulins (150 kDa), amino acid analysis may "underestimate" the total protein concentration. These results also show the effect of recovery of individual residues on protein quantitation. As expected, the recovery of more than one stable residue could be used to calculate total protein content of samples, which is in good agreement with the results obtained by Kjeldahl nitrogen assay. However, the protein concentrations calculated from the total mass of the recovered residues appear to give relatively low estimates in almost all cases. Thus, it is concluded that amino acid analysis is an appropriate reference method only when stable residues are employed for quantitation, particularly for highly purified proteins of rDNA origin.

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A new high-performance size-exclusion chromatography method has been developed for the determination of potency of human growth hormone products. This method has been extensively validated and shown to correlate well with the hypophysectomized rat bioassay which has been used traditionally. The method is much more precise than the traditional bioassay and thus provides more reliable means of producing consistent biosynthetic human growth hormone batches.

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An isocratic reversed-phase high-performance liquid chromatographic method for the determination of human growth hormone (HGH) purity is described. This method offers superior resolution of HGH-related substances (e.g., sulfoxide and desamido derivatives) from unmodified HGH when compared to a number of alternative chromatographic and electrophoretic techniques.

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Of patients who developed end-stage renal disease secondary to sickle cell anemia (SCA), some have undergone renal transplantation with reasonable success. We recently cared for a patient with SCA and a functioning, transplanted kidney who experienced a permanent decline in renal function three and one-half years following transplant. The evaluation of his renal dysfunction revealed multiple features to support recurrence of sickle cell nephropathy as the cause for the deterioration.

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An integrated approach is presented for the establishment of purified protein and peptide reference standard materials suitable for both biological and chemical assays. Preliminary considerations, handling and storage conditions, and a variety of chemical methods for defining the reference standards are examined in detail. Of the chemical methods, liquid chromatography (LC), amino acid analysis, Kjeldahl protein assay, electrophoresis, and ion chromatography are key assays in determining the potency, purity, and identity of the reference standard preparation. Finally, the role of liquid chromatography in assessing reference standards for identity and chemical purity is examined and correlated with other methods.

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A rapid and specific high-performance liquid chromatographic (HPLC) assay has been developed for the determination of enviradene, 1, at concentrations of 2-5 ng/mL in plasma. The drug was extracted from the samples using benzene. The benzene extract was evaporated and the residue dissolved in the mobile phase. The HPLC system consisted of a reversed-phase column and a 75% methanol:25% 0.2 M sodium acetate mobile phase. Either a UV detector set at 268 nm or an electrochemical (EC) detector set at a potential of +0.9 V (versus Ag/AgCl/3 M NaCl) was used to monitor the drug. A column-switching system was used to remove late-eluting plasma constituents that interfered in subsequent chromatograms. The limit of sensitivity was 2 ng/mL for the HPLC-EC procedure and 5 ng/mL for the HPLC-UV procedure. Recovery from plasma was approximately 97%; the procedure had a relative error of approximately 3% and a relative standard deviation of 4.5% over the range of 20-200 ng of 1/mL of plasma. Following intravenous administration of 1 or 2 mg/kg of 1 to dogs, the parent drug was quantitated in plasma for 24 h using this procedure. The terminal phase half-life in plasma was calculated to be 10 h. Oral administration to dogs of single 8 mg/kg doses of 1, formulated with povidone-30 or polysorbate 80 and microcrystalline cellulose, produced high and persistent plasma concentrations of drug. At doses below 2 mg/kg, plasma concentrations were found to be nonlinearly related to the amount of the dose administered. The bioavailability of the drug in dogs was found to be increased by the concomitant administration of food.

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We describe a sensitive, specific liquid-chromatographic determination of penbutolol and its 4-hydroxy metabolite in plasma and urine. The method involves a simple organic extraction, evaporation of the solvent, reconstitution in methanol/water, and injection into the chromatograph. Penbutolol, its metabolites, and the internal standard, propranolol, are resolved on a CN reversed-phase column and detected fluorometrically. Conjugates of penbutolol and its 4-hydroxy metabolite may be determined after a 2-h enzymic hydrolysis. Detection limits are in the range of 3 to 12 micrograms/L of plasma. The assay is reproducible and nearly free of interferences. Representative concentrations in blood and urine of normal volunteers are reported.

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Following cannulation of the right external jugular vein and the efferent duct of the right caudal mediastinal lymph node (the caudal end of this node having been ligated to cut off the inflow of systemic lymph), sheep were each given one of four "cephalosporins" (cefazolin, moxalactam, cefoperazone, or ceftriaxone) as single doses injected iv over 30 min. All of the drugs appeared in the pulmonary lymph during iv infusion. Peak concentrations in the lymph were attained at 5 min postinfusion with cefazolin, cefoperazone, and ceftriaxone; the peak for moxalactam was attained at 30 min postinfusion. Cefazolin and cefoperazone penetrated better than did ceftriaxone, which penetrated better than did moxalactam. The concentrations of moxalactam, as compared with the other drugs, declined more gradually in both venous blood and pulmonary lymph. In view of the prompt entry and transit through the lungs and the high concentrations attained in the pulmonary lymph, these drugs should be effective in the treatment of pneumonias caused by susceptible bacteria.

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Pharmacokinetic parameters were calculated from plasma and urine latamoxef ('Moxalactam') levels determined by HPLC assay after single and multiple intramuscular (im) and single intravenous (iv) doses of 500 mg given to eight healthy volunteers. After im administration, systemic bio-availability was 92% after both the first and sixth doses. Peak plasma concentration was 18 mg/l (first dose) and 22 mg/l (sixth dose), reached at 1.2 h and 1.3 h respectively. The terminal phase half-lives were 2.5 h and 2.7 h respectively. After iv administration, the initial phase plasma half-life was 0.23 h and the terminal phase half-life, 2.4 h. Plasma clearance was 87.0 ml/min. The steady state distribution volume was 210 ml/kg. After iv administration, 72% of the dose was found in the urine in the first 24 h. Urinary clearance was 66 ml/min (iv dose) and 63 ml/min (sixth im dose). Most systemic infections will permit eight hourly dosing with 500 mg im or iv. Many urinary infections will be best treated with im administration, rather than iv administration.

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A simple and specific method has been developed for determination of enviroxime in biological samples. Enviroxime, a substituted benzimidazole, its coisomer zinviroxime, and the internal standard hexestrol were extracted from the samples with benzene. The benzene layer was evaporated and the residue was reconstituted and injected onto a liquid chromatograph. Reversed-phase chromatography on an octylsilane column with a 65% methanol-35% 0.14 M sodium acetate mobile phase separated the components. The compounds were detected electrochemically using a glassy carbon electrode held at +0.85 V. The assay could detect as little as 4 ng of enviroxime/ml of plasma, 15 ng/ml of nasal wash, and 20 ng/ml of urine or tissue homogenate. For plasma assays, the procedure was greater than 97% accurate and had a relative standard deviation of less than 4%. This method has proven to be applicable and reliable for the determination of enviroxime in many types of biological samples. Several problems encountered during the routine use of electrochemical detection were explored and minimized.

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A high-pressure liquid chromatographic procedure was developed to determine the D and L isomers of moxalactam in human plasma and urine. After protein precipitation with hydrochloric acid the sample was extracted with ethyl acetate. It was then back extracted into tromethamine buffer (pH 8.0) and washed with octanol. Extraction recovery from plasma ranged from 73-81%. An aliquot of the tromethamine buffer was then injected onto a C18-muBondapak column. The mobile phase was 3% acetonitrile in 0.05 M ammonium acetate pH 6.5 buffer. Samples were quantitated by UV detection at 275 nm and 0.01 aufs. The lower limit of detection was 0.5 microgram/ml for each isomer. Preliminary stability studies were performed to assess proper sample handling and storage conditions. The procedure was evaluated in a clinical setting to demonstrate its applicability to the study of moxalactam pharmacokinetics in critically ill patients.

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Methimazole was administered orally and rectally in a single dose of 60 mg to six euthyroid volunteers (three females and three males). Blood levels of methimazole were the same whether administered by the oral or rectal route, with peak levels of 1184 +/- 118 and 1163 +/- 150 (+/-SEM) ng/ml respectively. This study provides evidence that the rectal administration of methimazole may be an alternative to treating hyperthyroid patients who are unable to take this drug by mouth.

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High-performance liquid chromatographic methods for determination of the isomers of moxalactam in plasma and urine have been developed. Conventional reverse-phase chromatography was used for plasma assays, and an ion-pairing reagent was included for urine assays. Detection limits were 1.5 micrograms/ml of plasma and 7.5 micrograms/ml of urine. The high-performance liquid chromatographic assays were extensively compared with a microbiological assay (detection limit, 1 microgram/ml), using samples from human volunteers to whom moxalactam had been administered as well as plasma and urine from untreated humans, to which moxalactam was added. The correlations between the assays were quite good, but the precision and accuracy of the high-performance liquid chromatographic methods were superior. Both types of assays were used in a study of the stability of moxalactam-containing samples at various temperatures.

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A simple and accurate method for measuring acetaminophen in serum was developed using liquid chromatography with electrochemical detection. Acetaminophen can be quantitated in 100 microliter of serum over the range of 20 ng/ml--20 microgram/ml. The method is linear (r = 0.9997) and reproducible (RSD = 3.0% at 2 microgram/ml, RSD = 5.1% at 200 ng/ml, n = 6). An internal standard (N-propionyl-p-aminophenol) is used, and a single extraction is performed.

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We have developed a system of computer programs to expedite analyses of amino acid data obtained in a clinical environment. The system contains a program for building and maintaining libraries of chromatogram data, and a program for retrieval of data on the basis of any of its associated biographical characteristics. Several programs have been written that work with data drawn from the libraries. They provide for easy presentation, manipulation, or statistical analysis of the data. Included are comparison of population means and variances and intra-population correlation analyses. A minicomputer that has 12K of core storage is adequate for use with the system.

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