Chemical methods in association with physical methods in drug analysis

129

trendsin analytical chemistry, vol. 4, no. 5,1985

Chemical methods in association with physical methods in drug analysis Klaus G. Florey NewBrunswick, NJ, U.S.A. Physical methods, namely spectroscopy and separation techniques such as high-performance liquid chromatography, in which the analyte is not chemically changed during analysis, have gained increasing popuhuity in the field of pharmaceutical analysis. Nevertheless, chemical manipulation in combination with such methods will continue to play a vital role, as will be demonstrated with examples selectedfrom methods deveLopedfor two new, cardioactive drugs, nadolol and captopril, andfrom other recent literature references.

Where does this leave chemistry of which Siggia claimed ten years ago that it will increase sensitivity, accuracy, precision, range of application and elimination of interferences? Let me illustrate the usefulness of chemical manipulation with some very relevant examples from our laboratories. They helped to solve analytical problems during the development of nadolol and captopril, two cardioactive drugs. In addition, I also shall present other examples selected from the literature.

Introduction ‘With the increase in our ability to make measurements, we have seen a tendency to decrease our use of chemistry for analytical purposes. In the days when the analytical chemist could just measure mass, volume and color, he relied heavily on chemistry to give him a measurable parameter to increase sensitivity, accuracy and precision, range of application of methods, to decrease analysis time, and to circumvent interferences. We should still use the chemical expertise and lines of reasoning used by our predecessors and apply this extra degree of freedom to all our modern measuring approaches’. Thus admonished the eminent analytical chemist Sidney Siggiai almost a decade ago. It is timely to explore whether Siggia’s eloquent message is still valid today where the use of powerful physical methods seems to be ever increasing. A classification of analytical methods into physical and chemical ones is somewhat arbitrary, but for the purpose of this presentation, I will define physical methods as those in which the analyte is not chemically changed during analysis. Such modern physical methods are absorption spectroscopy in the ultraviolet, visible and infrared regions, fluorescence and nuclear magnetic resonance spectroscopy and, particularly, separation techniques. Of these, high-performance liquid chromatography (HPLC) seems to be the current method of choice in pharmaceutical analysis. Indeed, in the Journal of Pharmaceutical Sciences of 1982,86% of all analytical publications described chromatographic methods and 58% HPLC methods, specificall y .

Nadolol Nadolol is a /3-adrenergic blocking agent. It is a mixture of two racemates (Fig. l), and it is important to determine the ratio of the two racemates which normally should be about 50:50.

01659936/85/$02.00.

OH

*I -CH~NHC(CHQ)~ HO

HO Each of four sets optically active

_ + +

+ + -

I

Racemate

A

I

Racemate

B

I

Both

optically inactive

Fig. I. Racemate composition of nadolol.

Each racemate has a different crystal form and, therefore, specific bands of infrared and powder Xray diffraction spectra could be identified and used to determine the ratio. However, we were apprehensive that possible polymorphism might obscure these measurements. A score of thin-layer and HPLC systems did not give the desired separation, but help came from a rather unexpected source. When the NMR characteristics of nadolol derivatives were explored, it was found that the tert.-butyl protons of the tetraben0 Elsevier Science Publishers B.V.

trendsin analyticalchemistry, vol. 4, no. 5 1985

130

R I

RO

RO

R= Tert. Butyl Protons: A:6 = 1.57 B:6 = 1.60

Accuracy

= f 2%

Fig. 2. Determination of racemate composition of nadolol by NMR.

zoate, but not those of nadolol itself, gave different signals (see Fig. 2) for racemates A and B. By measuring the ratios of the peak heights, the percent of racemate B could be calculated with an accuracy of about + 2%*. For best results, the tert.-butyl proton resonances were recorded three times on an expanded scale of 50 Hz and once on a scale of 250 Hz. This example then demonstrates that NMR solved a problem when measurement was preceded by derivatization. When given the assignment to develop a sensitive serum and urine level, Ivashkivs developed a fluoro-

/ (7

CH2,c

I

//

0

‘H

H2N

/

+

\

I H2N XI

CH2/iAH

R

\

metric method by taking advantage of the two vicinal hydroxyl groups in the molecule which, by a wellknown reaction, could be oxidized with periodate to the corresponding dialdehyde. Coupling of the dialdehyde with o-phenylene diamine in acid medium probably takes place and results in a fluorescing compound (excitation 305 nm; emission 445 nm) according to the scheme in Fig. 3. The method is very sensitive, since as little as 2 rig/ml of serum can be determined. Recently, this method was extended to HPLC?. The fluorescing derivative was chromatographed on a C,, column using acetonitrile-perchloric acid as mobile phase. Nadolol concentrations in human plasma were determined with about the same sensitivity and precision as with the Ivashkiv method. This example demonstrates that the use of chemical manipulation designed to induce fluorescence is worth consideration. The usefulness of gas-liquid chromatography (GLC) would be severely limited without the possibility of increasing volatility by derivatization. That certainly holds true for most compounds of medicinal interest, The literature on derivatization agents is extensive and will not be reviewed here. Under the premise on which this presentation is based, namely that physical methods are those in which the analyte is not chemically changed during analysis, mass spectrometry (MS) is a purely physical method only for the determination of the molecular ion. However, the great utility of MS resides in the fact that the molecule is fragmented, in other words changed chemically, and that these fragments give much useful information about its structure. In the electron-impact mode, the molecular ion is rarely very prominent. Therefore, when using mass spectrometry for quantitative measurement, selection of a fragment ion of sufficient abundance makes possible measurements in the nano- and picogram range.

R = trimethylsilyl

R = -0-CH2CHCH,NHCKH,), I OH Fig. 3. Fluorescing derivative of nadolol.

Nadolol N-methyl

nadolol

Rl = H

x = m/e

R1 = CH3

x = m/e 100

Fig. 4. Mass spectrometry fragmentation of nadolol.

86

131

trendsin analyticalchemistry, vol. 4, no. 5,1985

When a quadrupole mass spectrometer was interfaced with a gas chromatograph in our laboratories, it was only logical to attempt the determination of nadolol in serum and urine by selected ion monitorings. The electron-impact spectrum of the tri-(trimethylsilyl) ether derivative of nadolol yields a fragment of m/z 86 which represents 43% of the total ion current. This fragment ion is part of the side chain as shown in Fig. 4. The N-methyl analog of nadolol affording an ion of m/z 100 was chosen as internal standard. The sensitivity of the method is slightly better than the fluorometric one and gave equivalent results. It has become the method of choice in our laboratories. Captopril The next examples chosen concern the novel orally active antihypertensive drug, captopril6 (Fig. 5), which acts on the principle of inhibition of the angiotensin-converting enzyme.

0

COOH

Fig. 5. Captopril.

When we were called upon to develop a blood level method for captopril, we chose gas chromatography (GC)-selected-ion monitoring MS. Due to the labile sulfhydryl group, captopril is unstable in drawn blood. When added to whole blood or serum, it is oxidized to the disulfide at a rate of approximately l%/min. Chemical manipulation was needed to prevent the oxidation. N-Ethylmaleimide (NEM)7 was selected as the stabilizing agent because the Michael addition shown in Fig. 6 proceeds rapidly in aqueous solution. The purified NEM-captopril was esterified with methanolic hydrochloric acid and an aliquot was injected onto the GC-MS instrument. For ion monitoring, the m/z 230 ion shown in Fig. 7 was selected. The 4-fluoro-derivative of captopril giving a corresponding m/z 248 ion was used as internal standard. The m/z 230 and 248 ions are not the most abundant, however, they were chosen since they showed the least interference from other blood constituents. The sensitivity of the method is 5 rig/ml of bloods. It has served us well in analyzing thousands of blood samoles of natients. 1

I

0

vN

0

3

t

HS-R

-

vN SR a0

0

Fig. 6. NEM derivative of captopril (HS-R = captopril).

Since electrochemical detection coupled with HPLC involves either oxidation or reduction, it qualifies for discussion under the umbrella of our topic just as much as selected ion monitoring. It has been used to determine underivatized captopril in urine. Captopril, upon standing, also is oxidized in urine. However, it can be stabilized for long-term storage by acidification, trace metal removal and quick refrigerationg. The method for captopril was originally developed by Yehi in our laboratories, and it has been elaborated upon by Perrett and Druryii. Urine acidified with hydrochloric acid was directly injected onto an ODS-Hypersil column. The mobile phase consisted of an acidic, methanolic potassium phosphate solution. An electrochemical detector equipped with a gold/mercury cell was chosen. The cell was maintained at +0.07 V versuS the Ag/AgCl reference electrode, causing oxidation of the sulfhydryl group to the disulfide. The method was also adapted for the determination of captopril in blood which was centrifuged and deproteinized with sulfosalicylic acid. This example clearly shows that electrochemical detection coupled with HPLC is a tool which should be considered when sensitivity and selectivity are required. Tocainide In the case of nadolol, oxidation followed by coupling led to a product amenable to fluorometric determination. Fluorescence can also be induced by diR

N n,

Cl. OQ

230 248

N EM NEM

OCH,

128 146

captopril methylester R = H 4-fluoro-analog methyl ester

R=F

Fig. 7. Fragmentation of GLC derivatives of captopril: NEM captopril methyl ester (R = H) and NEM I-fluorocaptopril methyl es._ ter (R = F).

132

trends in analytical chemistry, vol. 4, no. 2 I985

FLUORESCAMINE

CH3

b 1

0

II NH-C-

;

H

I C-NH, CHa

-

CH3

TOCAINIDE

Fig. 8. Fluorescamine derivative of tocainide.

rect derivatization. I have selected a recent paper by Sedman and Gall2 in which the authors describe a serum method for tocainide which is coupled with fluorescamine, followed by HPLC with fluorometric detection (see Fig. 8). The publication provides an excellent concise discussion of the history and use of fluorescamine, a remarkable reagent which has found widespread use. Fluorescamine, non-fluorescent by itself, reacts with primary amines to give highly fluorescent pyrrolinone derivatives. Amoxicillin So far, in the examples involving HPLC, precolumn reactions were used. However, there is also a growing literature on post-column derivatization. In a recent publication, Freils discussed the advantages and disadvantages of pre- and post-column derivatization. He concluded that for pre-chromatographic techniques, straightforward as they might be, artifact formation might be a problem. On the other hand,

post-column reactions might offer advantages of sensitivity and selectivity, but the interdependence between the mobile phase and the reaction medium might pose a problem. A typical example of post-column derivatization described by Lee et al. 14 is the determination of amoxicillin in urine, again using fluorescamine as a reactant. Both amoxicillin and its penicilloic acid derivative (Fig. 9) could be determined simultaneously. A urine sample was diluted with aqueous methanol and an aliquot was injected into a reversed-phase column. Water-methanol-acetic acid was used as the mobile phase. Phosphate buffer was pumped into the effluent stream to adjust the pH. Then fluorescamine in acetone was metered in. After passing a delay coil, the effluent was sent through the spectrofluorometric detector. Obviously, the whole assembly is more complicated than ‘straight’ HPLC needing accurate metering from three pumps instead of only one. The sensitivity limit of the assay was described as 2.5-5.0 pug/ml of urine. Alkylene oxide polymers The final example has been in the literature for almost twenty years. Yet, it is worth mentioning, since it shows that one should not hesitate to use ‘radical chemical surgery,’ particularly in the analysis of Ethylene and propylene polymeric substances. oxide have been determined quantitatively as components of such substances as alkylene oxide polymer@ by hydrolytic bromination at elevated temperature followed by GLC of the resulting dibromides (Fig. 10) using flame ionization for detection. R

HBr 0-

CH,-

CH,

OR

150

“c

)

BrCH,

-

CH,Br

Fig. IO. Hydrolytic bromination of ethylene oxide polymers.

Recently, this method has been put to good use in our laboratories for the determination of Polysorbate 80 (Tween 80) in a radiopharmaceutical preparation. N”,

O=C-N-

CHCOOH.3H20 T

S CH-CC-NH-CH-CH

,C’-‘3

C, CH3

h,

I 6OOH

/‘I H” -

~HCOOH

II

Fig. 9. Amoxicillin (I) andpenicilloic acid derivative (II).

Conclusion I hope I have succeeded in demonstrating that chemistry continues to play a significant role in drug analysis. Indeed, it adds in Siggia’s words, ‘this extra degree of freedom’, to physical measurements which makes it possible to solve many otherwise intractable analytical problems. Let me paraphrase this as follows: The better chemist always will be the better analyst.

trend&

133

analytical chemistry, vol. 4, no. 5,1985

Acknowledgement This article is adapted and condensed from a chapter in D. D. Breimer and P. Speiser (Editors), Topits in Pharmaceutical Sciences, Elsevier, Amsterdam, 1983. References 1 S. Siggia, J. Chem. Education, 51(1974) 99. 2 M. S. Puar, The Squibb Institute for Medical Research, personal communication. 3 E. Ivashkiv, J. Pharm. Sci., 66 (1977) 1168. 4 J. Patel, D. Hoffman and T. Thomson, Abstracts, APhA Academy of Pharm. Sci., 12 (1982) 156. 5 P. T. Funke, M. F. Malley, E. Ivashkiv and A. I. Cohen, J. Pharm. Sci., 67 (1978) 653. 6 M. A. Ondetti, B. Rubin and D. W. Cushman, Science, 196

(1977) 441. 7 A. I. Cohen and K. J. Kripalani,

U. S. Pat., 4,179,568

(1979). 8 P. T. Funke, E. Ivashkiv, M. F. Malley and A. I. Cohen, Anal. Chem., 52 (1980) 1086. 9 H. Kadin and R. Poet, J. Pharm. Sci., 71(1982) 1134. 10 P. Yeh, presented at the Eastern Analytical Symposium, New York, NY, 1980, Abstract No. 60, p. 54. 11 D. Perrett and P. L. Drury, J. Liq. Chromatogr., 5 (1982) 97. 12 A. J. Sedman and J. Gal, J. Chromatogr., 232 (1982) 315. 13 R. W. Frei, J. Chromatogr., 165 (1979) 75.

14 T. L. Lee, L. D’Arconte and M. A. Brooks, J. Pharm. Sci., 68 (1979) 454. 15 A. Mathias and N. Mellor, Anal. Chem., 38 (1966) 472.. Klaus Florey was Director of the Analytical Research and Development Department of the Squibb Institute for Medical Research, New Brunswick, New Jersey, U.S.A. from 1959 to 1984. Atpresent, he serves as a scientific consultant to the Institute.

Notice to Advertisers TrAC offers a unique opportunity to advertisers to inform all those who use and develop analytical methods in research, industrial, government and clinical laboratories about new products and publications. TrAC has: * worldwide circulation * cut-out reader enquiry coupons for both the United States and the Rest of the World * special rates for series advertisement. For further information contact: Wilfeke van Cattenburch, Advertising Department, Elsevier Science Publishers, P.O. Box 211, 1000 AE Amsterdam, The Netherlands. Tel: 020 58 03 714i715 Telex: 18582 ESPA NL

TRILAB

CHROMATOGRAPHY DATA SYSTEMS

Channels - 10 Mbyte hard disc option *Softwarefor;GC-LC-HPLC-GPC-THERMALSIMULATED - DISTILLATION - QA etc @MainFrame Communications 04

@Lowcost entry point - Expandable to 16 enquiry terminals *Input from; Balances, Bar code readers, Laboratory instruments etc. *Main Frame Communications

Sunderland Road, Sandy, Bedfordshire SG19 lRB, England. Telephone: Sandy (0767) 82222. International+44 767 82222. Telex: 826478 TRISYS G.

v-

v Trivector Systems International Limited.

Circle no. 129 on reader enquiry form