ANALYTICAL CHEMISTRY, VOL. 50, NO. 2, FEBRUARY 1978
197
Fluorescence Photometric Quantitation of Procainamide and N-Acetylprocainamide in Plasma after Separation by Thin-Layer Chromatography Ram N. Gupta," Francis Eng, and Diane Lewis Department of Laboratory Medicine, St. Joseph's Hospital, McMaster University, Hamilton, Ontario, Canada
A thin-layer chromatographic method for the estimation of procalnamide and its major metabolite, N-acetyl-procainamide, In plasma is descrlbed. The method involves a plasma extraction at alkaline pH with dichloromethane to isolate the drug and Its metabolite. p-Amlno-N-(2-dipropylamlnoethyl)benramlde and Its N-acetyl derivative are added to the plasma prior to extraction as internal standards. A solvent system Is selected which allows excellent separation of the four compounds from each other; from plasma constituents and other drugs commonly prescribed with procalnamide. It Is demonstrated that densitometric scanning in the fluorescent mode Is a precise and specific technique to quantitate procalnamlde and N-acetylprocalnamldeseparated by thin-layer chromatography.
In the past, plasma procainamide (PA) levels have been determined in clinical laboratories as a guide to effective therapy (1). N-Acetylprocainamide (NAPA), the major metabolite of PA, also has a n antiarrythmic effect, with potency similar to t h a t of PA (2-4). T h e estimation of PA and NAPA is a more effective guide to therapy than the determination of P A concentration alone. Several methods for the simultaneous determination of PA and NAPA have been described. T h e simplest procedure t h a t can be carried out in any clinical laboratory is the colorimetric procedure ( 5 6 ) . In this technique, hydrolysis is used t o convert NAPA to PA. T h e concentration of P A in the extract is measured with and without hydrolysis using Bratton-Marshall reagent. T h e NAPA concentration is determined by the difference between t h e two measurements. This procedure lacks specificity. Benzodiazepines and their metabolites produce amines on hydrolysis which react with Bratton-Marshall reagent, producing t h e same purple color as PA. In the fluorometric procedure (7), the concentration of NAPA is measured at p H E 1 at ,A, = 288 nm, ,A, = 341 nm and t h e concentration of PA is measured a t alkaline p H a t A, = 298 and ,A, = 354 nm. This procedure also lacks specificity because other compounds that may fluoresce under the same conditions are not removed and it has a limited linear range. A number of gas chromatographic (GC) procedures have been described (2,8-11). All are more specific but are slow. T h e retention time for NAPA is more than 20 min. In these procedures, a single compound. similar in properties to those of PA, has been used as an internal standard. As a result, the quantitation of NAPA has not been ideal. A number of high performance liquid-chromatographic (HPLC) procedures have been described for the simultaneous estimation of PA and NAPA (12-14). T h e elution time for PA and NAPA in HPLC is much shorter than the elution time of these compounds in GC. However, it is also a relatively slow technique since only 0003-2700/78/0350-0197$01 .OO/O
one sample can be chromatographed a t a time. T o meet the increasing demands for drug analysis, we have opted to use thin-layer chromatographic (TLC) techniques wherever possible. T h e instrument used for quantitation, a T L C spectrophotometric scanning system, is capable of estimating quantitatively a number of preseparated samples relatively rapidly. Spectrophotometric and fluorometric modes are available to quantitate the separated compounds by reflectance, by transmittance, or by simultaneous reflectance and transmittance. In addition, the separated compounds on T L C plates can be chemically converted to colored or fluorescent products, an ability that is not generally available in the commercial H P L C instruments. A TLC procedure for the estimation of PA and NAPA has been described ( 3 ) . T h e quantitation has been carried out by measuring t h e quenching of a fluorescent indicator incorporated into the TLC plate by the separated compounds. This technique has been shown to be erroneous (15). More recently, a TLC procedure has been published where PA and NAPA have been estimated by UV reflectance (16). We describe a T L C procedure where PA and NAPA and their corresponding internal standards are separated 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 mode with a mercury vapor lamp as the light source for excitation and a Zeiss No. M365 filter for isolation of the emitted light. Both a strip-chart recorder (Linear Instruments Corp., Irvine, Calif.) model 355, 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. M. Merck, No. 5763) were developed in TLC tanks with twin chambers (Terochem Laboratories, Edmonton, Alberta). Reagents. All reagents were of analytical grade and were used without further purification unless otherwise specified. Procainamide Stock Standard. Procainamide hydrochloride was purchased from Pfaltz and Bauer, Inc., Flushing, N.Y. A stock solution of PA (1 mg/mL) was prepared by dissolving 57.7 mg of PA.HC1 in 50 mL of methanol. N-Acetylprocainamide Stock Standard. N-Acetylprocainamide was prepared by treating 57.7 mg of PA.HC1 with 100 pL of acetic anhydride and 200 pL of redistilled pyridine. The mixture was placed in a boiling water bath for 2 h, dried in a current of dry nitrogen, and dissolved in 50 mL of methanol to give a concentration of NAPA 1 mg/mL. No trace of unreacted PA was detected when 10 wg of the acetylated product was chromatographed and sprayed with Bratton-Marshall reagent sequential spraying of TLC plate with: 20% (v/v) sulfuric acid; 1% (w/v) sodium nitrite; 5% (w/v) ammonium sulfamate and 1% methanolic solution of N-1naphthylethylenediamine dihydrochloride. Under similar conditions, 10 ng of PA produced an intense purple spot. p-Amino-N-(2-dipropylaminoethyl)bentamide(BA)Stock Standard. BAaHCl was kindly supplied by E. R. Squibb & Sons, Inc., Princeton, N.J. A stock solution of BA (1 mg/mL) was C 1978 American Chemical Society
198
ANALYTICAL CHEMISTRY, VOL. 50, NO. 2, FEBRUARY 1978
Table I. R f Values of PA, NAPA, BA, and NABA
R f values
Solvent systems
PA
NAPA
BA
NABA
1.Benzene:ethyl acetate :ethanol :ammonia (15 :15: 5 :1) 2. Benzene:ethyl acetate:methanol :ammonia (160 :80: 1 6 0 : l ) 3. Benzene:ammonia:l,4-dioxane(10: 5 :80) 4. Isopropanol :chloroform :ammonia (45: 45: 2) 5. Benzene:ammonia:acetone:dioxane(10 :4: 60: 20) 6. Benzene:ethyl acetate:methanol:ammonia (20:lO:20: 1) 7. Ethyl acetate :methanol :ammonia ( 4 2 :5 : 2 ) 8. Isopropanol :chloroform :ammonia ( 4 5 :45 :5 ) 9. Ethyl acetate:methanol :ammonia (88: 8 : 4 )
0.38 0.26 0.54 0.45 0.70 0.70 0.52 0.67 0.32
0.29 0.26 0.42 0.52 0.67 0.71 0.42 0.73 0.23
0.64 0.64 0.80 0.87 0.92 0.88 0.80 0.92 0.74
0.51 0.70 0.71 0.88 0.87 0.87 0.66 0.94 0.62
of the solvent flow using the Zeiss Chromatogram scanner. The scanner was used in the fluorescence (M-PR) mode (,lex = 313 nm and M365 filter for emission; slit length = 10 mm and slit width = 0.3 mm). The emission peaks were recorded on the strip chart recorder and also simultaneously integrated by the electronic integrator. Under these conditions, peaks due only to PA and BA and the solvent front were observed. B. The same TLC plate was then placed into the equilibrated dry chamber of the twin chamber developing tank. The other chamber contained 10 mL of concentrated hydrochloric acid. The plate was exposed to the acid for 15 min and then exposed to air for 5 min and then rescanned (Aex = 297 nm and M365 emission filter; slit length = 10 mm and slit width = 1.5 mm). The only emission peaks observed were due to NAPA, NABA, and the solvent front. The plasma PA or NAPA concentrations were determined from standard curves established by plotting peak area ratios of standard/internal standard (PA/BA or NAPAINABA) against PA and NAPA concentrations of the standards.
prepared by dissolving 29 mg of BA.HC1 in 25 mL of methanol. N-Acetyl-p-amino-N-(2-dipropylaminoethyl) benzamide (NABA) Stock Standard. NABA (1 mg/mL) was prepared and checked as for NAPA stock standard. The stock solutions were stored a t 4 "C and were stable for at least three months. Plasma S t a n d a r d s . Combined plasma standards containing 5,10,15, and 20 mg/L of both PA and NAPA were prepared by adding appropriate volumes of the stock (1 mg/mL) solutions of PA and NAPA to drug free pooled plasma. The plasma standards were divided into 2-mL aliquots and frozen until assay. Internal S t a n d a r d . A combined internal standard solution containing 10 mg/L BA and 10 mg/L NABA was prepared on the day of analysis by diluting appropriate volumes of the stock solutions of these compounds with dichloromethane. P a t i e n t S a m p l e s . Venous blood from patients receiving procainamide therapy was drawn 1 h after the last dose into evacuated heparinized blood collection tubes. Plasma was separated within 2 h of collection and stored at 4 "C until analyzed. The samples were frozen when they were to be stored for more than 24 h. Procedure. E x t r a c t i o n . To 0.5 mL of spiked plasma (standards) or patient plasma in 16 X 125 mm glass culture tubes with Teflon-lined screw caps, 0.5 mL of internal standard solution, 0.1 mL of 1 M sodium hydroxide and 5 mL of dichloromethane were added. The tubes were shaken on a mechanical shaker for 2 min. Approximately 5 g of anhydrous sodium sulfate was added to each tube and the tubes were again shaken for 2 min and centrifuged. The dichloromethane layer from each tube was decanted into correspondingly labeled 16 x 100 mm glass test tubes and evaporated in a water bath at 45-50 "C. The residue was dissolved in 100 pL of methanol. Chromatography. Methanol residue, 1 FL from each tube was spotted 15 mm apart on a TLC plate. The plate was developed in solvent No. 9 (Table I) to a height of about 15 cm and air dried for 10 min. The residues of the standard plasmas were spotted on each plate if more than one was chromatographed. Detection. A. The plate was then scanned along the direction
RESULTS AND DISCUSSION Recovery from plasma spiked with PA, NAPA, BA, and NABA a t a concentration of 10 mg/L was determined by comparing each extracted compound with the area of corresponding amounts of methanolic standards spotted directly. The recoveries varied from 88--98%. During extraction there is no change in the ratio of PA and its internal standard, BA, or NAPA and its internal standard, NABA, when mixed in ratios of 0.5 to 5 . The addition of internal standards (BA and NABA) prior to extraction to all plasmas (standards and patient sera) obviates the need of a quantitative procedfie for extraction and for precise spotting of the residue on TLC plates. T h e use of internal standards compensates for extraction irregularities or unforeseen losses.
I
I.A.
B.
C.
i
'I ' I
SOLVENT
SOLVE
I I
NAPA
j
Figure 1. Chromatograms of PA, BA, NAPA, and NABA. (A) Fluorescent mode; alkaline pH, A,, = 313 nm, A,,, = 365 nm, slit width 0.3mm. = 365 nrn, slit width 1.5 mm. (C) UV reflectance mode; alkaline pH, X = 265 nm, slit width (8) Fluorescent mode: acidic pH, A,, = 297, ,A, 0.5 mm.
ANALYTICAL CHEMISTRY, VOL. 50, NO. 2, FEBRUARY 1978
Table 11. Estimation of Precision A. Within-batch variation PA Mean ISD N RSD
2.0Q 0.08 9
3.7%
NAPA
6.4Q 0.10
10.5Q 2.Oa 0.14 0.09
6.4Q
9.gQ
0.21
0.24
10
10
8
10
9
1.5%
1.3%
4.5%
3.2%
2.5%
B. Between-batch variation Mean ISD N RSD
PA
NAPA
5.4Q 0.085
5.4Q
10
1.6%
0.182 10 3.4%
mg/L. Table 111. R f Values by Fluorescent Detection before or after Exposure t o HCl
R f value
PA NAPA BA NABA 1. Caffeine 2. Carbamazepine 3. Chloropromazine 4. Codeine 5. Diazepam 6. Diphenhydramine 7. Flurazepam 8. Isoniazid 9. Librium 10. Lidocaine 11. Meprobamate 12. Methaqualone 13. Methylprylon 14. Phenozuximide 15. Primidone 16. Propanalol 1 7 . Quinidine Sulfate 18. Theophylline
0.32 0.23 0.74 0.62
-
-
-
0.51 0.42
-
199
response. T h e baseline is still quite stable (Figure 1). T h e procedure is linear from 1 mg/L to 30 mg/L of PA and NAPA (RSD of PA = &2.7%, N = 6, and RSD of NAPA = 1 1 . 2 % , N = 6). T h e precision of this procedure for both PA and NAPA is excellent. Within-batch precision was tested with plasma samples between 2 and 10 mg/L. Between-batch variation was tested using a plasma a t a level of 5 mg/L for both PA and NAPA in ten consecutive runs (Table 11). Throughout this investigation, only one lot of pre-coated silica gel plates was used. The incorporation of a standard curve on each plate chromatographed in this technique should eliminate problems encountered with minor deviations in plates. T h e plasma samples in the procedure are subjected to a simple dichloromethane extraction a t an alkaline pH. A number of neutral and basic compounds other than PA, B.4, NAPA, and NABA could also be extracted if present in the plasma. As shown in Table 111, some of the drugs which may be administered along with procainamide do not interfere. Fluorescence adds to the specificity of the thin-layer chromatographic procedure. T h e need to scan the TLC plate twice for the quantitation of PA and NAPA in this procedure is time consuming: however, we have not been able to find conditions where both the compounds could be quantitated simultaneously by fluorodensitometry. T h e separated compounds can be quantitated simultaneously by UV reflectance either before or after exposure to acid vapors. One internal standard is adequate for the quantitation of PA and NAPA by this method. However, in the UV reflectance mode, the baseline is not as stable as in the fluorometric mode (Figure 1). T h e procedure is very sensitive. There is no problem distinguishing plasma containing 0.5 m g / L of PA or NAPA from a blank plasma. If required, as little as 50 pL of plasma can be assayed. T h e amounts of internal standards are correspondingly decreased and the residue of the extracts are reconstituted in smaller volumes of methanol. We conclude that the quantitative TLC procedure described here for PA and NAPA is an acceptable routine method.
LITERATURE CITED Plasma standards containing known amounts of PA and NAPA are processed with each plate to obtain a standard curve. T h e spotting of methanolic standards has been found to be unnecessary. A number of solvent systems were tried for the separation of PA, NAPA, BA, and NABA. T h e results are summarized in Table I. T h e solvent system selected: ethyl acetate: methano1:ammonia (88:8:4), allows optimal separation of these compounds and shows minimal edge effect. It has been shown t h a t procainamide has good native fluorescence (Aex = 298 nm, ,A, = 354 nm) a t p H 11.0 and N-acetyl procainamide has fair native fluorescence (Aex = 288 nm, , ,A = 341 nm) a t p H 1.0 (7). The internal standards, BA and NABA, behave similarly to PA and NAPA in their fluorescence characteristics. Since the developing solvent contains ammonia, the p H of the TLC plate, even after drying, is optimal for fluorescence photometry of PA and BA. T h e baseline is steady and essentially noise free (Figure l ) , and PA and BA show excellent response. After exposure t o acid, NAPA and NABA become fluorescent; however, the fluorescence exhibited is less than that for the same concentration of PA or BA, therefore the slit width is increased from 0.3 to 1.5 m m to achieve greater
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RECEIVED for review August 9, 1977. Accepted November 21, 1977.
