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graphite tube, t h e pyrolytic coating is eroded a n d some reduction in sensitivity results. T h e sensitivity of the silica test varies somewhat from day t o day due to variation in the quality of the pyrolytic coating on t h e graphite tube. T h e properties of t h e pyrolytically coated tube are more important for t h e silica test, in which charring and atomizing temperatures are somewhat higher t h a n for many other FAAS tests. Because a 1OO-gL sample is required for the trace silica test, it is important to carefully control t h e drying time and temperature t o avoid loss of sample by boiling during t h e drying stage. A drying time of 80 s was chosen t o ensure proper drying of t h e sample even if t h e drying temperature needs t o be set slightly less t h a n 100 OC. A lower drying temperature may be required when the autosampling device is used for an extended period of time for sample injection, since t h e graphite atomizer is not cooled as efficiently after numerous sample analyses as in the initial sample injections. T h e analyst must monitor t h e drying stage after about 10 sample analyses t o make certain t h e sample is slowly evaporating, not boiling during t h e drying stage. Sometimes a spurious peak is observed immediately following t h e silica atomization peak. Since t h e highest atomization peak within t h e preset integration time (usually 4 s) is recorded by the spectrophotometer, the integration time may need t o be set between 3 and 4 s to assure t h a t only the silica atomization signal is measured. T h e wavelength must be carefully adjusted on the spec-
455
trophotometer t o properly set t h e wavelength a t 251.6 nm. There are several other sensitive wavelengths in t h e same region - 250.7, 252.4, and 252.8 nm. Improper wavelength adjustment will result in loss of sensitivity. Care must be exercised t o avoid silica contamination. No glassware should be used in the preparation of silica standard solutions or samples. All receptacles, e.g., such as polyethylene pipet tips, polystyrene beakers, a n d 2.0-mL cups for t h e automatic sampling device, must be pre-rinsed with either low-silica water and/or t h e solution t h e receptacle is t o contain. The flush water used for the AS-1 autosampler must be low-silica water. Samples requiring trace silica analysis should be collected in high density polyethylene bottles.
LITERATURE CITED (1) J. T. Woods and M. G. Mellon, I&. Erg. Chem., Anal. M . 13,760 (1941). (2) American Public Heab Association "Standard Methods fw the Examination of Water and Wastewater", 14th ed.,1975, p 490. (3) J. C. Guillaumin, At. Absorpt. News/., 13, 135 (1974). (4) H. L. Kahn, M. Bancroft, and R. H. Ernrnel, Res.lDev., 23 (7), 30 (1976). (5) K. C. Thompson, R. G. Godden, and D. R. Thornerson, Anel. Chlm. Acta, 74, 289 (1975). (6) David 8. Lo and Gary D. Christian, Can. J. Spectrosc., 22, 45 (1977). (7) H. M. Ortner and E. Kantuscher, Talanta. 22, 581 (1975). (8) P. Lagas, Anal. Chim. Acta. 98, 261 (1978). (9) "Encyclopedia of Industrial Chemical Analysis", Snell and Ettre, Ed., Interscience Publishers, New York 1973, pp 18, 39. (10) Joseph L. Dlnnin, Anal. Chem., 32, 1475 (1960). (11) J. 8 . Willls. Spectrochim. Acta. 16, 259 (1960).
RECEIVED for review November 9, 1978. Accepted December 21, 1978.
Fluorescence Photometric Determination of Disopyramide and Mono-A/-dealkylated Disopyramide in Plasma after Separation by Thin-Layer Chromatography R a m N. Gupta," Francis Eng, and Diane Lewis Department of Laboratory Medicine, St. Joseph ' s Hospital, Hamilton, Ontario, Canada
Cyrus Kumana Department of Medicine, McMaster University, Hamilton, Ontario, Canada
A thin-layer chromatographic method for the estimation of disopyramide and its metabolite, mono-N-dealkylated disopyramide is described. The method involves a simple extraction of 200 pL of plasma at alkaline pH with benzene. An aliquot of the extract is quantitatively spotted on a TLC plate. The plate is developed in the solvent (ethyl acetate:methanokammonia, 40:30:0.5) which allows good separation of the two compounds from one another, from plasma constituents, and from other drugs which are prescribed along with disopyramide. The plate is dipped in 20 YO sulfuric acid in methanol to make these compounds fluorescent. Densttometric scanning of the separated compounds provides a precise quantitative estimatlon. The procedure is linear up to 10 mg/L and the calibration curve passes through the origin.
Disopyramide (DIS) is an anti-arrhythmic drug effective in t h e treatment of a broad spectrum of atrio-ventricular arrhythmias ( I ) , a n d has recently been introduced in North America. I t is metabolized primarily to mono-N-dealkylated disopyramide (MND) ( 2 ) ,which probably has less therapeutic 0003-2700/79/0351-0455$01 .OO/O
activity than DIS. In most instances, it has been the practice t o correlate clinical effect solely with plasma concentrations of the parent drug, but for several reasons knowledge of plasma MND concentrations may also be useful. If treatment failure is associated with low DIS levels, high plasma M N D levels would favor rapid metabolism as the cause rather t h a n noncompliance. Data on the plasma concentration effect of t h e MND metabolite is still relatively meagre in the clinical setting and the MND metabolite, although less active than DIS, could be important in patients having a high proportion of metabolite; particularly if the dosage of DIS administered is being titrated to yield a given plasma concentration. Individual differences in sensitivity may exist between patients both for plasma concentration of DIS and/or MND. In long-term therapy, the metabolizer status of the patient could prove helpful where side effects emerge. Initially, DIS was evaluated by a spectrofluorometric procedure ( 3 ) . This procedure measures both the parent drug a n d its metabolite as both of them are nearly equally fluorescent in the presence of strong acid (Aex = 275 nm; An = 410 nm). For clinical use, this procedure lacks specificity 0 1979
American Chemical Society
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as other compounds present may produce fluorescence under the experimental conditions.
R = -CH(CH,),
= DISOPYRAMIDE
R = H = MONO-N- DEALKYLATED DISOPYRAMIDE
In the past few years, gas chromatographic procedures have been described using either a flame ionization detector (4-7) or a nitrogen-selective detector (8). However, only one of these procedures ( 4 ) describes the simultaneous estimation of the parent drug and its metabolite. T h e sample preparation of this procedure is complex, tedious, and not suitable for a routine laboratory. T o overcome the problems of analysis of these compounds by gas chromatography, high performance liquid chromatographic procedures using paired-ion techniques for the simultaneous estimation of DIS and MND have been described (9). After separation, these compounds are estimated by their UV absorption a t 254-258 nm. We describe a procedure in which DIS and MND are separated by TLC and then estimated by fluorometric densitometry.
EXPERIMENTAL Instrumentation. A TLC scanning spectrophotometer, model KM3 (Carl Zeiss, Canada Ltd., Don Mills, Ontario) was used in the fluorescent (M-Pr) mode with a mercury vapor lamp as the light source for excitation. A Turner filter, No. 110-812,narrow pass, 405 nm (Fisher Scientific Co., Toronto) with a holder improvised to fit the Zeiss scanner was used for isolation of the emitted light. It was inconvenient to use Zeiss M-405 filter in M-Pr mode. Both a strip-chart recorder, model 355 (Linear Instrument Corp., Irvine, Calif.) and an Autolab System IV computing integrator (Spectra Physics, Santa Clara, Calif.) were used for calculation. Silica gel 60 glass plates, 20 X 20 cm, without fluorescent indicator (E. Merck, No. 5763) were developed in TLC tanks with twin chambers (Terochem Laboratories, Edmonton, Alberta). The extracts were applied to the TLC plates using a TLC multispotter equipped with 250-yL syringes and a nitrogen flow system (Analytical Instrument Specialties, Libertyville, Ill.). A dipping tank (Whatman Inc., Clifton, N.J.) was used to acidify the plate. All volume measurements were made with SMI micro-pettors (Scientific Manufacturing Industries, Emeryville, Calif.). Reagents. All reagents were of analytical grade and were used without further purification. Disopyramide, chlordisopyramide and mono-N-dealkylated disopyramide were obtained as gifts from Searle Canada Ltd., Oakville, Ontario. Stock standards (1mg/mL) of each of these compounds were prepared by dissolving appropriate amounts of the hydrochloride salts in 0.1 N HCl. Plasma Standards. Combined plasma standards containing 2.5, 5, 7 . 5 , and 10 mg/L of both DIS and MND were prepared by adding appropriate volumes of the stock standards of DIS and MND to drug-free pooled plasma. The plasma standards were divided into 2-mL aliquots and frozen until assay. Patients' Samples. One hour after the last dose, venous blood from patients receiving regular disopyramide therapy was drawn into evacuated, sterile, heparinized, blood collection tubes. Plasma was separated within 2 h of collection and stored a t 4 "C until analyzed. The samples were frozen when they were to be stored for more than 48 h. Procedure. To 0.2 mL of spiked plasma standards or patient plasma in 13 X 100 mm glass culture tubes, with Teflon-lined screw caps, 50 FL of 6 N NHIOH and 1mL of benzene were added. The tubes were shaken on a mechanical shaker at low speed for
5 min and were then centrifuged. Before loading the 250-pL syringes onto the TLC multispotter, each syringe was rinsed with a benzene extract and then filled accurately to the 100-pL mark with the same extract. The spotter heater was maintained at 50-60 "C in a gentle atmosphere of nitrogen. The plunger drive was driven at medium speed to complete the spotting in about 5 min. The extracts of all standards were spotted on each plate if more than one plate was used. The plate was developed in solvent, ethyl acetate/methanol/ammonia (40:30:0.5),to a height of about 12 cm and air-dried for 10 min. The plate was then dipped in 20% sulfuric acid in methanol. The back of the plate was wiped dry with tissue paper and allowed to stand in air in subdued light for 45 min. The plate was then scanned in the fluorescence mode (Aex = 266 nm, A,, = 405 nm; slit length = 10 mm, and slit width = 2 mm). The emission peaks were recorded on the strip chart recorder and simultaneously integrated by the electronic integrator. Under these conditions, peaks only due to DIS, MND, and the solvent front were observed. The plasma DIS and MND levels were determined from standard curves established by plotting peak areas of the standards against the DIS and MND concentrations of the standards.
RESULTS AND DISCUSSION I t is common practice to use internal standards for the assay of drugs in body fluids by chromatographic procedures t o obviate the need of accurate measurements a t various steps. p-Chlorodisopyramide (Cl-DIS) has been used as internal standard in GC ( 4 ) and HPLC procedures (9). In our initial experiments, we also used this compound as an internal standard, which was added t o plasma prior to extraction. I t was found that the intensity of fluorescence of C1-DIS was about 5 times that of DIS. Further, it proved difficult to select a suitable solvent for ideal separation of the three compounds from each other and from other fluorescent anti-arrhythmic drugs, particularly quinidine. The procedure described in this report is simple and does not involve many volume measurements and evaporation of extracts. When the assay of DIS and MND without the addition of an internal standard was attempted, better precision overall was achieved than that found with the initial procedure including the internal standard. I t was decided t o use a solvent which would provide the upper layer to facilitate the collection of extracts. A number of solvents less dense than water were tested for extraction efficiency and for ease of spotting and benzene was found t o be the most suitable solvent. Recovery from plasma spiked with DIS and M N D was found to be about 90%. This was determined by comparing the peak areas of DIS and MND in extracted plasma standards to the peak areas of unextracted methanolic standards of the same concentration. T h e extraction efficiency is unaffected by change in p H between the range of 9-13. T o minimize exposure to benzene vapor, spotting is carried out in the fume hood. In the present procedure, an auto-spotter is used for convenience and speed. However, it is not essential to do so. Plasma (0.2 mL) can be extracted with less volume (0.2 mL) of benzene and 20 yL can be spotted manually in a current of nitrogen without loss of precision. However, it is tedious to do so when a large number of samples are to be spotted. Disopyramide has been estimated fluorometrically in the presence of strong acid with the excitation a t 275 nm ( 3 ) . In the present procedure, high fluorescence was observed when A,, = 266 nm was used. However, the mercury line a t 266 nm is weak and requires a wide slit width (1.5-2 mm). T h e base line is still quite stable under these conditions (Figure 1). Different concentrations of aqueous and alcoholic solutions of sulfuric acid, perchloric acid, and phosphoric acid were compared for the development of optimum fluorescence of disopyramide and its metabolite. T h e reagent used in the present procedure, 20% methanolic sulfuric acid, gave the best
ANALYTICAL CHEMISTRY, VOL. 51, NO. 3, MARCH 1979 c
Table 11. Blind Sample Study
0
E c
5 3 P
lA
457
concn of spiked samples, wg/mL
B
no. of samples
0.5 1.0 2.0 2.5
2 2 2 2 4
2.75
6
3.0 3.5 8.0
2 2 3
1.8
-
observed values 0.45, 0.45 0.76, 0.84 1.92, 2.08 2.12, 2.17 2.52, 2.73 2.42, 2.62 2.88, 2.86 2.81, 2.64 2.91, 2.92 3.50, 3.34 3.47, 3.50 7.01, 7.18 7.10
DIRECTION OF SCAN
Figure 1. Chromatogram of extracts: (A) of plasma spiked with 4 mg/L each of DIS and MND; (B) of plasma from a patient receiving 4 X 200 mg/day of DIS. Fluorescence mode: A,, = 266 nm, ,A, = 405 nm, slit width = 2 mm
Table 111. Interference Studies
compound
Table I. Estimation of Precision
mean, mg/L 1 S.D., mg/L N C.V., 7i mean, mg/L 1 S.D., mg/L N C.V., %
2.11
2.02 0.04 10 2.4
4.01 0.10 10 2.6
MND
5.94 2.02 0.11 0 . 1 3 0.12 10 10 10 10 5.3 3.1 2.2 5.8 B. Between-batch variation 5.90 0.18
2.04 0.08
10
10
3.1
4.0
4.01 0.07 10 2.0
6.02 0.12 10 2.0
4.01 0.17 10 4.4
6.01 0.11 10 1.9
0.40
DI S MND C1-DIS caffeine codeine chlordiazepoxide diazepam diphenylhydantoin flurazepam lidocaine procainamide propranalol quinidine
A. Within-batch variation DIS 4.09 0.13
concn Rfa mdL of solvent solvent plasma 1 2 0.51 0.39
0.20 0.48 20 5 10 5 40 5
lo lo
5 10
0.58
__ __ -__ -----
__ -__ -__ -___ --
0.57
0.67
--
Solvent 1 = ethyl acetate/methanol/ammonia (40:30:0.5). Solvent 2 = methanol/ammonia (100:1.5). ( - -) = Not detected; stays at origin or migrates with solvent front. a
results. Initially, the fluorescence continues to increase while t h e plate is drying a n d then after 3 h there is a gradual decrease in fluorescence. The fluorescence of MND is about 80% t h a t of DIS. The procedure is linear to 10 mg/L for both DIS and MND a n d t h e standard curve passes through t h e origin. When a test sample shows a level greater t h a n 10 m g / L , T L C quantitation is repeated using 50 pL of the extract. T h e precision of this procedure for both DIS and MND is satisfactory when compared to current methodology ( 5 , 9). Within-batch and between-batch precision was tested with plasma samples spiked with different concentrations of these compounds within t h e therapeutic range (2, 4,and 6 mg/L) a n d t h e results are summarized in Table I. Twenty-five blind serum samples, spiked with DIS by G. D. Searle and Company, Oakville, Ontario, were submitted for analysis by the T L C procedure. T h e data are presented in Table 11. After evaluation, the TLC method was considered t o be acceptable for t h e analysis of DIS in plasma. A number of plasma constituents a n d other drugs, if present, could also be extracted due to t h e simple benzene extraction employed. Therefore, plasma spiked with a number of drugs which may be administered with disopyramide was carried through the procedure. As shown in Table 111, these drugs do not interfere with t h e assay of disopyramide or its metabolite. Fluorescence adds to t h e specificity of the thin-layer chromatographic procedure. Chromatographic procedures are not absolutely specific a n d it is not possible within t h e scope of this study t o exclude all potential interferences. I n t h e last six months, we have analyzed more t h a n 50 samples from patients receiving disopyramide and we have not observed any interference (distorted peaks or
Table IV. Steady-State Plasma Concentrations ( h e - d o s e level, Le., 6 hours post-dose)
patient 1
2 3 4 5
6
6 hourly dose, mg
DIS, mg/L
MND, mg/L
DIS/MND
200 200 200 150 150 100
1.8 4.9 3.0 0.6 2.9 1.7
1.8 0.3 1.2 2.3 0.4 0.1
1.0 16.3 2.5 0.7 7.3 17.0
unusually high values for DIS or MND). If an interference is suspected, TLC would be repeated using an alternate solvent z2: methanol/ammonia (1OO:l.S). When more t h a n one chromatographic system is employed, the combined discriminating power increases ( I O ) . As shown in Table IV, there is a wide variation in the plasma levels of DIS and M N D in patients receiving similar doses of disopyramide a n d in the ratio of MND/DIS. The procedure is sensitive. DIS or MND can be quantitated a t a concentration of 0.5 m g / L of plasma. T h e quantitative TLC procedure described here for the estimation of DIS and MND is an acceptable, efficient method for incorporation into a routine laboratory.
ACKNOWLEDGMENT We are indebted t o F. Louman, G. D. Searle & Co., for providing pure compounds and spiked samples.
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LITERATURE CITED (1) L. A. Vismara, D. T. Mason, and E. A. Amsterdam, Clln. pharmacal. Ther., 16, 330 (1974). (2) A. Karim, R. E. Ranney. and S.Kraychy, J . Pharm. Sci.. 61, 888 (1972). (3) R. E. Ranney, R. R. Dean, A. Karim, and F. M. Radzialowski. Arch. Int. Pharmacodyn., 191, 162 (1971). (4) T. C. Hutsell and S. J. Stachelski, J . Chromatogr., 106, 151 (1975). (5) J. W. Daniel and S. Subramanian, J. Int. Med. Res. Suppl. 1 , 4, 2 (1976). (6) A. Johnston and D. McHaffie, J . Chromatogr., 152, 501 (1978).
(7) A. M. Hayler and R . J. Flanagan, J . Chromatogr., 153, 461 (1978). (8) A. M. J. A. Duchateau, F. W. H. M. Merkus, and E. Schobben, J . Chromatogr., 109, 432 (1975). (9) P. J. Meffin, S. R. Harapat, and D. C. Harrison, J . Chromatogr., 132, 503 (1977). (10) A. C . Moffat and K. W. Smalldon, J . Chromatogr., 90, 9 (1974).
RECEIVED for review September 15,1978. Accepted December 9, 1978.
CORRESPONDENCE Improved Timing Resolution in Time-Correlated Photon Counting Spectrometry with a Static Crossed-Field Photomultiplier Sir: Time-correlated photon counting is a power analytical technique for studying a variety of properties of chemical systems. Recently, reported experimental measurements using gated detection techniques have an overall timing uncertainty (light source plus detection system) of 1 ns or less, limited principally by the transit time dispersion in conventional photomultiplier tubes (1-4). Because many physiochemical processes of interest display a temporal behavior requiring time-correlated measurements for analysis and/or to improve signal-to-noise ratio, even small reductions in timing uncertainty on the subnanosecond time scale can extend significantly the applicability of this analytical method to, inter alia (1) the study of fluorescent molecules with very short lifetimes, (2) the elimination of Raman background-scattering from fluorescence emission spectra, (3) time-resolved emission spectra of transient species, and (4) the enhancement of Raman spectra by rejection of fluorescence background emission. Previously, we reported the use of a synchronously pumped tunable dye laser producing very short light pulses (
