Analytical Methodology in
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The development of assay procedures for new drugs pro vides a challenge for the analytical chemist, especially when his problems involve the detection and measurement of drug concentrations in the blood plasma, tissues, and ex creta of laboratory animals and human subjects. T h e assay procedures must be highly specific and sensitive, often ex tending down into the nanogram range. Many advances in this area have been due to the efforts of chemists and bio chemists whose achievements have gone unrecognized be cause of the urgency in going from one problem to another, without taking adequate time to write up the results for publication. Years ago I decided that we would continue to publish regularly, at least in terms of the most significant advances in our laboratory, even if it meant much personal time and effort to do so. I believe every one of us has an obligation to the scientific community to publish the results of his work, and fortunately we had the full support of manage ment in this endeavor. However, over the years during the development of analytical methodology, I became aware of a progressive continuity that is not often evident from the usual scientific publications. One purpose of this presenta tion is to describe the background leading to the develop ment of some novel analytical techniques within our labo ratory. The drug metabolism group at Parke-Davis was estab lished about 30 years ago by Dr. A. Calvin Bratton, Jr., who came to Detroit to head the Pharmacology Department. H e was trained as a physician and pharmacologist at Johns Hopkins and had an excellent background in organic chem istry. While working with Dr. E. K. Marshall, Jr., at Balti more, he became interested in analytical techniques for drugs and synthesized N-(l-naphthyl)ethylenediamine dihydrochloride, which is still used as a coupling reagent for diazonium compounds today (2) (see Figure 1). Although the idea of measuring the plasma levels of drugs was still in its infancy, Dr. Bratton recognized the positive relationship of drug plasma levels to therapeutic effect and established the first drug metabolism laboratory in the pharmaceutical industry. His wisdom and foresight have been fully confirmed. Even though he is now retired, his patient guidance will be long remembered. In 1947, with a nucleus of three trained individuals, we started developing analytical procedures for drugs that were under investigation at that time. Our basic responsi 632 A · ANALYTICAL CHEMISTRY, VOL. 50, NO. 7, JUNE 1978
bility was to examine the effect of laboratory animals and human subjects upon the drug under investigation. This is quite distinct from the approach used by the pharmacol ogist and toxicologist in studying the effect of the drug upon the intact animal. One of the more interesting compounds being studied at that time was a newly discovered antibiotic that had been isolated from fermentation beers and was known to have an aromatic nitro group in its structure (2).
This was later known as Chloromycetin or chloramphenicol. Being well acquainted with the Bratton-Marshall reagent for aromatic amines, we turned our attention toward a quantitative reduction procedure to convert the nitro group to a primary amine. Our first effort to heat acidified solutions of the drug with zinc dust produced a diazotizable amine that could be coupled with the Bratton-Marshall re-
Figure 1 . Bratton-Marshall c o l o r i m e t r i c r e a c t i o n f o r primary a r o m a t i c a m i n e s involving diazotization w i t h nitrous a c i d , elimination of e x c e s s nitrous acid by r e a c t i o n w i t h s u l f a m i c acid, a n d coupling of diazonium salt w i t h Bratton-Marshall reagent ( / ) , A/-(1-naphthyl)ethylenediamine dihydrochloride 0003-2700/78/0350-632A$01.00/0 © 1978 American Chemical Society
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agent to yield a violet color. We could get quantitative re covery from plasma specimens with an analytical sensitivi ty that could detect less than 1 μg/mL (3). This involved the preparation of a protein-free filtrate, reduction by heating with zinc dust in acid solution, filtration, prepara tion of a diazonium salt by treatment with nitrous acid, re moval of excess nitrous acid by the addition of ammonium sulfamate, and finally coupling with the Bratton-Marshall reagent. There was little interference from normal body constituents, but some interference was found with adenine derivatives and folic acid. As a sidelight, we worked up the same procedure for the assay of adenine, which yielded an orange-colored complex with the Bratton-Marshall reagent (4). Since the zinc reduction step required an extended peri od of heating, with inevitable evaporation losses and vol ume changes, we looked for other reducing agents that could be used at room temperature. These ranged from stannous chloride to hypophosphites, hyposulfites, alkaline glucose, and even electrolytic reduction, but the best re agent was titanous chloride, which acted very rapidly at room temperature (3). Unfortunately, the excess reagent had to be removed by precipitation as a hydrated oxide in alkaline solution, followed by centrifugation, or it would in terfere with diazotization. An aliquot of the supernatant solution was then acidified and run through the usual JCO\orimetric assay procedure. This basic procedure has been used widely in clinical work to determine plasma levels in patients receiving chloramphenicol, and it showed excel lent agreement with microbiological assays for the antibiot ic. In the mid-1960's, Wesley Dill, my colleague in many of these experiments, improved this procedure by the addi tion of sodium fluoride before the reduction step. The tianous chloride reagent formed a complex with fluoride ions. This did not interfere with subsequent diazotization and coupling—all steps were carried out in a single testtube cuvette. This technique was applied to a study of vari ous generic formulations of chloramphenicol (5), demon strating for the first time wide differences in the absorption of this drug when improperly formulated (Figures 2 and 3). More recently, Wesley Dill has employed fluorescamine for the assay of the primary aromatic amine of chlorampheni col, resulting in greatly improved sensitivity. On a drop of finger blood, we are now able to run quantitative assays
Metabolism Studies
Figure 2. Mean plasma levels of chloramphenicol in groups of 10 normal adult human subjects receiving 0.5-g oral doses of four different commercial formulations of chloramphenicol Reprinted with permission from ref. 5. Copyright 1968 C. V. Mosby Co.
Figure 3. Mean urinary excretion rate and recovery of aro matic nitro compounds in groups of 10 subjects receiving 0.5-g oral doses of four different commercial formulations of chloramphenicol ANALYTICAL CHEMISTRY, VOL. 50, NO. 7, JUNE 1978 · 633 A
with a sensitivity and specificity more than adequate for monitoring clinical subjects. Following the early work on chloramphenicol, we attempted to extend the same technique to other aromatic compounds by quantitative nitration of the phenyl ring on a micro scale, followed by reduction to a primary amine, diazotization, and coupling with the Bratton-Marshall reagent to yield a colored product. This concept was applied successfully to the assay of Dilantin (diphenylhydantoin or phenytoin).
Interestingly, the nitration will not work with large crystals of phenytoin, but it works very well indeed when the drug is extracted with an organic solvent and then evaporated to dryness at the bottom of a test tube. In other words, the rate of nitration is dependent in part on the surface area of the microcrystalline drug in contact with the nitrating reagent. The assay procedure was published by Dill et al. in 1956 (6) and represented the first satisfactory assay procedure for clinical use. Some interference was encountered with phénobarbital, which is often administered with Dilantin. This was eliminated by a three-plate countercurrent extraction step prior to nitration. By use of this technique, our study of the metabolic disposition of this drug in laboratory animals and in man led to a better understanding of drug action. In fact, the colorimetric procedure was used extensively in clinical laboratories throughout the world until the advent of gas chromatography (GC). Fortunately, we made an early start in GC assays by use of a Barber-Coleman Model 10 chromatograph with an argon ionization detector, followed rather quickly with a hydrogen flame ionization detector. The first GC assay procedure for Dilantin was developed in our drug metabolism group by Tsun Chang (7, 8) (Figure 4). This was based upon extraction of plasma with an organic solvent and formation of a trimethylsilyl derivative, which proved to be unstable in the presence of moisture and required special precautions to ensure quantitation. The same technique proved to be suitable for assay of the major metabolite of Dilantin, a parahydroxylated derivative (p-HPPH). The real contribution of Tsun Chang was in the selection of the p-tolyl analog of Dilantin (MPPH) for use as the internal standard (7). It has virtually the same extraction characteristics as Dilantin and is used in most clinical laboratories today for GC assays of Dilantin. At the time of this work, m-HPPH or the metahydroxylated derivative of Dilantin was proposed as an internal standard for p-HPPH assays. However, it was later shown by Atkinson et al. that this derivative was formed as a normal metabolite of Dilantin in the dog (9), and traces were found in acid hydrolyzed human urine. Chang et al. (10) then went on to isolate and identify a dihydrodiol metabolite of Dilantin in human urine, which, upon heating in acid, formed equal amounts of the meta- and parahydroxyphenyl metabolites. He also was reponsible for the identification of the 3,4-dihydroxyphenyl metabolite and the 3-methoxy-4-hydroxyphenyl metabolite in human urine (11, 12). We were still not satisfied with Dilantin assay procedures because of the time consumed in GC assays, and pos6 3 4 A « A N A L Y T I C A L CHEMISTRY, V O L . 5 0 , N O . 7, JUNE
1978
Figure 4. Gas chromatogram of trimethylsilyl derivatives of phenytoin (DPH) and its primary metabolite, 5-(p-hydroxyphenyl)-5-phenylhydantoin (HPPH) Also shown are phénobarbital (PB), the internal standard 5-
