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5-1-1981 Simultaneous high performance liquid chromatographic determination of procainamide, N-acetylprocainamide, disopyramide, mono-N- dealkyldisopyramide, quinidine, and propranolol in serum James F. Wesley
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Recommended Citation Wesley, James F., "Simultaneous high performance liquid chromatographic determination of procainamide, N-acetylprocainamide, disopyramide, mono-N-dealkyldisopyramide, quinidine, and propranolol in serum" (1981). Thesis. Rochester Institute of Technology. Accessed from
This Thesis is brought to you for free and open access by the Thesis/Dissertation Collections at RIT Scholar Works. It has been accepted for inclusion in Theses by an authorized administrator of RIT Scholar Works. For more information, please contact [email protected]. SIMULTANEOUS HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC DETERMINATION
OF
PROCAINAMIDE, N-ACETYLPROCAINAMIDE, DISOPYRAMIDE,
MONO-N-DEALKYLDISOPYRAMIDE, QUINIDINE, AND PROPRANOLOL
IN SERUM
James F. Wesley
May 1981
Submitted as part of the requirements
for a Master of Science Degree in
Clinical Chemistry
at
Rochester Institute of Technology
Rochester, New York 14623
Department of Clinical Sciences PERMISSION FOR REPRODUCTION
Simultaneous high performance liquid chromatographic determination of procainamide, N-acetyl procainamide, disopyramide, mono-N-dealkyldiso- pyramide, quinidine, and propranolol in serum. I, James F. Wesley, hereby grant permission to the Wallace Memorial Library of R.I.T., to reproduce the document titled above in whole or in part. Any reproduc tion will not be for commercial use or profit. TABLE OF CONTENTS
Table of Figures 2
Table of Tables 4
Abstract 5
Introduction 6
Experimental 8
Apparatus 8
Reagents 8
Procedure 9
Chromatographic Conditions 10 Linearity 10 Precision 13
Interferences 23
Correlation 38 Time/Cost Study 48 Method Development 51
Mobile Phase Development 51
Method Optimization 67 Drug Extraction 77 Conclusion 82
Reference List 83 TABLE OF FIGURES
Fig. 1. Chromatograph of Components Assay 11 Fig. 2. Procainamide Calibration Curve 14 Fig. 3. N-Acetylprocainamide Calibration Curve 15 Fig. 4. Disopyramide Calibration Curve ,g Fig. 5. Mono-N-dealkyldisopyramide Calibration Curve ,7 Fig. 6. Quinidine Calibration Curve 18 Fig. 7. Dihydroquinidine Calibration Curve 19 Fig. 8. Propranolol Calibration Curve 20 Fig. 9. Chromatogram of Serum Extracts 28 Fig. 10 Quinidine Correlation 42
Fig. 11 Procainamide Correlation ..
Fig. 12 N-Acetyl procainamide Correlation .,-
Fig. 13 Disopyramide Correlation 7 Fig. 14 Effect of Mobile Phase Methanol /Acetonitrile Ratio 52 on Drug Elution Fig. 15 Chromatogram of Assay Components at a Mobile Phase 54 Organic Ratio of 100% Methanol /0% Acetonitrile
Fig. 16 Chromatogram of Assay Components at a Mobile Phase 55 Organic Ratio of 0% Methanol/100% Acetonitrile
Fig. 17 Chromatogram of Assay Components at a Mobile Phase 56 Organic Ratio of 60% Methanol /40% Acetonitrile
Fig. 18 Effect of Mobile Phase Phosphate Buffer pH on Drug 57 Elution
Fig. 19. Effect of Mobile Phase Phosphate Concentration on Drug 59 Elution
Fig. 20. Effect of Mobile Phase Flowrate on Drug Elution 61 Fig. 21. Drug Resolution Equation 62 Fig. 22. Effect of Column Temperature on Drug Elution 63 (Flowrate 1.0 ml /min)
Fig. 23. Effect of Column Temperature on Drug Elution 64 (Flowrate 0.5 ml /min) Fig. 24. Isocratic Elution 66
Fig. 25. Gradient Elution; System A 68
Fig. 26. Gradient Elution; System B 69
Fig. 27. Gradient Elution; System C 70
Fig. 28. Gradient Elution; System D 71 Fig. 29. Gradient Elution; System E 72 Fig. 30. Gradient Elution; System F 73
Fig. 31. Liquid Chromatographic UV Absorbance Scan of Propranolol 75 TABLE OF TABLES
Table 1. Detector Linearity at 212 nm 12
Table 2. Linear Regression Analysis of Calibration Curves 21
Table 3. Extraction Efficiencies 22
Table 4. Within-Run Precision Data 24
Table 5. Run-to-Run Precision Data 25
Table 6. Summation of Precision Data and Ratios of Drug/ 26 Internal Standard
Table 7. Run-to-Run Retention Time Variation 27
Table 8. Interference Study; Drugs Tested at Their Indicated 29 Concentrations
Table 9. Interference Study; Drug Elution and Peak Area 30 Table 10. Interference Study; Extraction Efficiencies 32
Table 11. Interference of Glutethimide and Carbamazepine with 34 Propranolol
Table 12. Interference Study; Effect of Lipemic, Icteric, and 36 Hemolyzed Sera
Table 13. Interference Study; Suitability of EDTA and Heparinized 37 Plasma
Table 14. Thin Layer Chromatographic Identification of Dihydro- 39 quinidine
Table 15. Quinidine Correlation 41
Table 16. Procainamide/N-Acetylprocainamide Correlation 43
Table 17. Disopyramide Correlation 46 Table 18. Time Study 49 Table 19. Cost Study 50 Table 20. Ultraviolet Absorbance Scan Data 76
Table 21. Comparison of Extraction Efficiencies Using Differ- 80 ent Organic Solvents ABSTRACT
We describe a method for the simultaneous high performance liquid chromatographic determination of several antiarrhythmic drugs and some of their metabolites after extraction from 2.5 ml of spiked pooled sera.
The extracts were applied to a C8 reversed phase column. Nine compounds of interest were resolved within the 30 minute run. An initial mobile phase of 80% phosphate (25 mmol/L, pH 3.5), 20 % organic (acetonitrile: methanol, 2:3) was maintained for 2 min at which time a linear gradient was used to change the mobile phase to 30% phosphate, 70% organic at
20 min after injection. This composition was maintained for an addi tional 5 min. Absorbance at 212 nm was used for detection. Peak area ratios of drug to internal standard (N-propionylprocainamide) were used for quantitation. The relative standard deviations (and mean solute concentrations) of daily duplicate determinations for 15 days are: procainamide (PA), 5.1% (5.9 mg/L); N-acetylprocainamide (NAPA), 9.3%
(6.0 mg/L); mono-N-deal kyldisopyramide (NDAD), 3.7% (4.1 mg/L); diso pyramide (DIS0P), 4.3% (4.0 mg/L); quinidine (QUIN), 4.5% (6.5 mg/L); propranolol (PR0PL), 5.1% (97 yg/L); and dihydroquinidine (DIHYQ), 9.3%
(0.69 mg/L). A propranolol metabolite, 4-hydroxypropranolol (4-OHP), was resolved but not quantitated. INTRODUCTION
Quinidine, procainamide, disopyramide, and propranolol are drugs that are prescribed for their cardiac antiarrhythmic qualities. The determination of the blood serum concentration of each drug may be use ful to the physician in an effort to improve the effectivenes of ther apy.
Quinidine is a myocardial depressant (as are procainamide and di sopyramide) that may cause serious side effects at serum concentrations that are only ten percent higher than the therapeutic range. These include life threatening arrhythmias, serious hypotension, and hyper sensitivity reactions (1).
Procainamide usage in certain patients, characterized as "slow acetylators", may produce a potentially fatal syndrome similar to Lupus
Erythematosus. This condition has been related to the rate of conver sion of procainamide to its pharmacologically active, acetylated meta bolite, N-acetyl procainamide. Nausea and weakness may occur at pro cainamide serum levels fifteen percent higher than the therapeutic range.
Serum concentrations of N-acetyl procainamide are not known when these toxic symptoms appear (1).
Disopyramide may cause worsening arrhythmia and electrocardiogram changes with high doses and increased serum concentrations. Mono-N- dealkyl disopyramide is a major metabolite whose pharmacoactivity is still undetermined (1).
Propranolol is a 3-adrenergic blocking agent which affects the cardiac action of the catecholamines. There is poor correlation of drug dosage with blood concentration that is believed to be due to large variations in the rate of metabolism (1). Serum levels are usu ally requested to ensure that a minimum therapeutic concentration has been achieved and/or if the patient is in compliance with therapy.
Side effects at high levels may rarely include congestive heart failure
4- and pulmonary edema. One of the fifteen identified metabolites,
possess antiarrhythmic hydroxypropranolol , is believed to activity (2).
Our objectives in developing a high performance liquid chromato graphic method for the quantitation of procainamide, N-acetyl procainamide, disopyramide, mono-N-dealkyldisopyramide. quinidine, and propranolol were threefold. First, we sought to develop an assay for these drugs that would be free from major interferences that are present in the fluorometric methods for quinidine (3) and propranolol (4) and the colorimetric method for procainamide (5). Secondly, we wanted a means of detecting and quantitating serum levels of the major drug metabolites, whether or not they are reported to have pharmacologic activity, to aid in drug dosage regimens. Lastly, we needed a method that could be
used for the simultaneous determination of as many of the antiarrhythmic drugs and metabolites as possible. In this way, we could best utilize a microprocessor controlled liquid chromatograph with an autosampling
system that could run unattended and perform an analysis on an extracted
specimen regardless of which drug or combination of drugs was present
in the sample.
Miller and Tacker have reviewed previous HPLC methods for multi-
component serum analysis (6). To our knowledge, this is the first
report of a method that can be used for the simultaneous measurement
of several antiarrhythmic drugs and metabolites in serum. EXPERIMENTAL
APPARATUS
A Hewlett-Packard Model 1084B high performance liquid chromato graph was used in this study. It is a microprocessor controlled system in which keyboard entries are used to control several parameters; including a linear, variable by time, elution gradient; mobile phase flow rate; column temperature; and detector wavelength selection. The microprocessor also integrates peak areas by continuously monitoring the slope of the chromatogram. The sample injector is a variable volume syringe that can be operator adjusted to deliver 10 to 200 yL.
An automatic sampling system allows up to 60 samples to be run without further operator intervention. The variable wavelength detector is a single cell, dual wavelength type with an operating range of 190 nm to
600 nm.
An Altex Ultrasphere Octyl C8 (25 cm x 4.6 mm i.d.) column (Rainen
Instrument Co., Woburn, MA 01801) was used for the separations. A
Brownlee RP8 guard column (Rainen Instrument Co., Woburn, MA 01801) was used to prolong the analytical column lifetime.
Clin-Elute CE-1003 columns (Analytichem International, Inc., Lawn- dale, CA 90260) were used to extract the drugs and internal standard from serum.
Silica Gel 60 (0.2 mm thickness) pre-coated thin layer chromto- graphy sheets (EM Reagents, American Scientific Products, McGaw Park,
IL 60085) were used as the TLC stationary phase.
REAGENTS
All aqueous solutions were prepared with deionized water that had been passed through a 0.22 y filter. Acetonitrile and methanol were
HPLC grade (Fisher Scientific Co., Pittsburgh, PA) and used without further purification. Reagent grade H3P04, NaH2P04'H20, concentrated
NH40H, and ethyl acetate (Fisher Scientific Co.) were used as received.
The drugs, metabolites, and internal standard, used to prepare stock methanol solutions, were not purified further. Quinine, N-acetyl procainamide (both from Sigma Chemical Co., St. Louis, M0 63178), and N-propionyl procainamide (Pierce Chemical Co., Rockford, IL) were
8 purchased. The following were gifts supplied by the respective manu facturers: procainamide- HC1 (E.R. Squibb & Sons, Princeton, NJ 08540); disopyramide phosphate and mono-N-dealkyl disopyramide (G.D. Searle and
Co., Chicage, IL 60680); propranolol *HC1 and 4-hydroxypropranolol (Ayerst Laboratories, Inc., New York, NY); quinidine-S04 (Eli Lilly and Co., Indianapolis, IN 46206); and dihydroquinidine (A.H. Robbins, Co., Richmond, VA 23220).
Stock solutions were prepared in calibrated conical centrifuge tubes by dissolving each substance in an appropriate amount of HPLC grade methanol (approximately 5 mL) to obtain a concentration of 1.0 g of free drug/L.
Working methanol stock solutions were prepared for N-propionyl- procainamide and propranolol of 0.2 g/L, respectively, by appropriate dilution of the concentrated stocks. To preserve the integrity of the 4-hydroxypropranolol, which is unstable for long periods in methanol,
60 yL of stock solution was transferred to several test tubes, the methanol evaporated under N2 at 50 C, and the residue was stored stop pered at room temperature (60 yg per tube).
PROCEDURE
The aliquots of frozen pooled sera (standards, control, and blank are thawed at room temperature and vortexed for 10s to ensure homogen- iety.
Pipet 200 yL of 0.1 mol/L HC1 into labeled 16 x 150 mm glass col lection tubes that are then positioned below a corresponding Clin-Elute column. The pH of the column is adjusted by adding 200 yL of 1 mol/L Na?C0^. This is followed by the addition of internal standard, 50 yL of N-propionyl procainamide (0.2 g/L in methanol). Pipette 2.5 mL of serum onto the column and wait 3 min for equilibration. Three additions of 4 mL each of the extraction solvent (methylene chloride:hexane:isoamyl alcohol, 49:49:2, v:v) follow. Wait for the dripping to stop before
collection for and cen adding the next portion. Vortex the tubes 45s trifuge at 2000 rpm for 10 min. Aspirate and discard the organic su pernatant. Add 300 yL of methanol to the remaining aqueous phase in 50 C the collection tube, vortex for 5s, and evaporate to dryness at under a gentle stream of N2 Dissolve the residue in 150 yL of the initial mobile phase (described below) and inject 75 yL into the liquid chromatograph.
CHROMATOGRAPHIC CONDITIONS
The mobile phase was in two reservoirs: one contained 25 mmol/L phosphate, pH 3.5, and the other, a mixture of acetonitrile and methanol
(2:3). The phosphate was prepared by mixing 25 mmol/L solutions of H3P04 and NaH2P04 until a pH of 3.5 was achieved. This aqueous solution was then passed through a 1.2 y Millipore filter under vacuum which also effectively de-gassed the solution. The organic mixture was de gassed by heat and vacuum in the chromatograph reservoir.
30 The elution was performed at C with a flow rate of 1.0 mL/min.
The initial mobile phase composition was 80% phosphate and 20% organic.
At 2 min after the injection of sample, a linear gradient was started that reached 30% aqueous and 70% organic after 20 min into the run.
This mixture was maintained for another 5 min when the run was termin ated. The mobile phase was returned to its initial composition and allowed to equilibrate with the column for 5 min before another sample was chromatographed. Total run time was therefore 30 min.
The variable wavelength detector, with a 12 yL, 10 mm flow cell, was set at 212 nm for sample monitoring. The reference wavelength was
430 nm. A Chromatogram of all assay components is presented in Figure 1.
LINEARITY
The linearity of detector response, as a function of the concentra tion of each component of interest, was confirmed by comparison of the peak areas of two-fold serially diluted mixtures in mobile phase. Table
1 lists the range of each substance for which the area did not deviate more than 10% from the expected dilution factor. The serum concentra tions that would correspond to the measured linearity range by use of our extraction procedure, assuming 100% recovery, are also shown. The accepted therapeutic ranges are included for comparative purposes.
The 4-hydroxypropranolol proved to be unstable in methanol (i.e. stock solutions) and in mobile phase. The stability of a fresh solution was sufficient to determine the chromatographic retention time, but it was not adequate for quantification. This propranolol metabolite is reported to have amphoteric extraction characteristics (7), and a serum concentration of 5-40 yg/L (8). It was concluded from this
10 1.0
UJ o z < OD OC O CO A B CD <
25 MINUTES
Fig. 1. Chromatogram of antiarrhythmic drugs, metabolites, and internal standard. The components (1.5 yg each) are identified by their respec tive retention times, are: A, procainamide (5.68 min); B, N-acetylpro- cainamide (7.64 min); C, N-propionyl procainamide (10.88 min); D, mono- N-dealkyldisopyramide (14.90 min); E, 4-hydroxypropranolol (15.94 min); F, disopyramide (17.51 min); G, quinidine (18.51 min); H, dihydroquinidine (19.79 min); and I, propranolol (21.49 min).
11 TABLE 1. DETECTOR LINEARITY AT 212 nm.
Equivalent Serum Therapeutic Serum Amount Concentration3 Concentration0 Substance Injected (yq) (mq/L) (ma/L)
Procainamide 0.27 - 17.5 0.22 - 14.0 4 - 8
NAPA 0.14 - 17.5 0.11 - 14.0 4 - 8
NPP 0.14 - 17.5 b -
NDAD 0.03 - 17.5 0.03 - 14.0 ?
Disopyramide 0.07 - 17.5 0.05 - 14.0 2 - 4
Quinidine 0.14 - 17.5 0.11 - 14.0 3 - 6
210d 100d Propranolol 0.005 - 0.33 3 - 20 -
a Assumes 100 percent recovery in reported extraction procedure (see Table 3 for actual recoveries). b Internal standard. c From reference (1). d Concentration yg/L.
12 information that 4-hydroxypropranolol was an inappropriate compound to include in this assay.
Linearity of the overall assay procedure was determined through the use of calibration curves. These were prepared from spiked sera with the use of N-propionyl procainamide as the internal standard. The peak area ratio of drug /internal standard (ordinate) was plotted against the concentration of drug in the serum spike (abscissa). The high standard is approximately twice the high therapeutic serum concentration in each case. Serial dilutions of the high standard to concentrations of 1/3 and 1/9, using the same serum pool but without drug or metabolite as the diluent, were prepared to cover the full range of clinical in terest. Standard curves for all drugs and metabolites are shown in
Figures 2-8. Linear regression analysis data of the standard curves are summarized in Table 2. The small values for the Y-intercept indi cates that no endogenous interferences are present. This was confirmed by extraction and elution of blank sera.
Absolute recovery was studied by the comparison of peak areas from stock solution residues, that had been reconsituted in mobile phase, with the peak areas from extracts of a spiked serum pool. Extraction efficiencies for several serum concentrations of each drug are summar ized in Table 3. The internal standard (N-propionyl procainamide) had an absolute recovery of 82% (SD t 4% n=12) when 50 yL of a 0.2 g/L methanol solution was added to the Clin-Elute column as described in the procedure. Although the absolute recoveries of N-acetyl procainamide and propranolol are less than 100%, they are also independent of con centration and therefore should be adequate for clinical use.
PRECISION
The reproducability of the injection system of the chromatograph was determined by injecting pure drug standards (n=10). The variation in retention times for any analyte was always less than 0.1 min. The relative standard deviation of each chromatographic peak area was less than 4% for all of the drugs except for the 4-hydroxypropranolol. When peak area of each drug was divided by the area of the internal standard, relative standard deviations improved to less than 1%.
Precision studies of drug quantification were conducted with two
run-to- pools of spiked sera, one for within-run variation and one for
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EEEll Eiiii: . __jl Xi 1 iif.EHE-E ^ = 1 " i- _ ._....Z- ... 4 4.. i .._ . : j he ~V*H [ - : EX . , j .EEEE -I [i , ~XE/TX X iEli : =t EEiE.E| if iiii; !E \Z- - l- =zzz.zX: oo f eE_ A 1 1 ZZ i TEXLff "El .. IfET^^XfX azaaz'z _1 _ . " - EEEE ZZZZXZZZZ _i ""E/'X EEIEE, ieeEie C CO i-iiii 1 p. _.. -- - - 1 E*T z. , -i T oc i 1 ! . . 1 i_- EE __ 1 "-- - E 1 [ I t.._L IE! ~-~yy\z: -- EEEX - i , i - 7.EEE7 1 1 XeeX r -. 1 yA X"': _ , EEiEl i . j :. - _ -_ : . y ZZZZZ i . ill . y -EEE1 i u- zzzzz EiE EE. ^B 1.1- 1 eX EEXEXX. EifJE^ UJ r E_Eil-i Q. i-fltfE.. 1 1: i-E y* E_._i.ii : 1 UJ j__ ___. . i TET :i" AAAAzz-^ x z x :.e *0 o ~ - - - o. 1 r __J -E>X z_ ee_. he . 4 ; TE! Hi , - s^~ z: -1 y- I i i J . -^-p:=1 y^^~ XI o- , ii::: 1 , - - 4 _11E EEE---- "_1 ' - 1 r El..J EE eeEei 2.0 4.0 6.0 8.0 10.0 12.0 QUINIDINE CONCENTRATION mg/L Fig. 6. Quinidine calibration curve from spiked serum extracts. Comparison of peak area drug/peak area internal standard with drug concentration in mg/L. Each point represents an average of three determinations. Standards are: 12.0 mg/L, 4.0 mg/L, and 1.3 mg/L. 18 ^ I : EX :: E Hid .... . zz.z XX if-^iE -Id . I. Az.z xt f z: '. .'.px: -Ed|Ei:.EE - 4 - J. ETXE x: Ei- 1 _ -":;-. f If [iiii - - En _._.. ___ 1-111 XEfX ;:rEX EX" 1 -- - _ -llE. : EEE-E i .-i--- X-.-: - EfEfJEX !Ei EH! EE-E. \_. -:if 7 - - i-i j ZZZ" -1 i .ZZZZ lliE IE X ZZZZZ HE! HE _ E . iiE-.i 7: .1 : _:::... - EJHE- ---!- 1. EETE-P XX -- 1 f EEXE|EEEEl] _ - _t 'J" ._...!) HE. E "' AZ H ] " " " E"_>. -_E - - . iTEXEE EEp. H . i 1 : . '^Ll -.l-l!' - - zzzz . - 4 IE! " :.- _. .i ... ET .1 1 4 1 H f x.r-.-E ::f ]f ill El - -7-i - i. _ E r_ - ... IEEE i i _ . zzzz- i- f-- "- - T 1- ; _ n.i.J liiii E- ] z -XEf f-0- "-, -::-( -H-r "| - --_:: . - i -In i-'."HE 1 'l-H ...-zl . . E ":0 --__i- ...:..-! "'I ElHE.E ..,._( 1 _. , - - -EitEE . E - " :'r-...~ j.- 1_ --"_. : -1 1 IE 1 et ... XEE iXEEEEE 1 HTE! !E , -u- X" 4 . - 1 Eil - _ . I EEE i .- x zz^z . i XIEXE ' - -7 _. _. 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I_ J 1 + -l-i-E-i - . f ill t" .... i.:e.e ... . ~A VT Xfjf. EE! - . r XE EX i_H! zzzz EEEH- XXE zzzz. ::|EiE , _ . . f,. . . ------1- _ ! i 1 , Z'ZZf. 1 j i:q ' EhX^e . . ... ;_ Xff xijxif__ _ u_ ,- :.. _ Q_ ro j \ - - -U 1 _.__ -..-. __-.. . _ UJ -_..| " - Hid O - - EEfEX X^>1 _ -A- .- - i x i. _i -J --E: X-- - - _ .._... - - ..J. . ^^EEEE , EEfEEI ._ 1 1 - _. -. - 1 [._._ 1 1 . ^EPX^it EEEEilE! .Hi---"iii hi! : "zzz. OS 1 1 i - - EfjE- - -1i _l - ..-Zzxaa-a: . :n_EI XErf+XXEE ZzZl'~zz E < X EEXTEXEE ' - 1" " CM L _ .... i 1 4- - - - -- 111 ' - - " Ei r. ^Lizz.zzz 1_| H < ' 4 : EE4_ IE ._... . _ . _J -.. v. 4^ r LU !"" 4_ . i ; , !-- - - - _ __X . ! , Q. HE i EH. ] ^ EXE7X xxnif | i . 1 : ^T- _ UJ -zzzzz. , Eli E -EX ^s. i P t-zf::_-... J L_ - ^ t -H EEill l-H yz \ -T_.-- .zzzEE eeXie . _,. - u. o- O o - i cc i --'-(_X ------. EiEJnEi P - - Q ^E i ~i i H j. _ ... - i .. . _ E : 1 Eiil ----- --^:.zjz~zzzzzzzzzz ^y.~ i L- +zz - - - E- . EX E -f. EEEl" -^EXei . zzzzzzzzz: -EE EEf : EH [n-Xdil 0.2 0.4 0.6 0.8 1.0 1.2 DIHYDROQUINIDINE CONCENTRATION mg/L Fig. 7. Dihydroquinidine calibration curve from spiked serum extracts. Comparison of peak area drug/peak area internal standard with drug con centration in mg/L. Each point represents an average of three determin ations. Standards are: 1.2 mg/L, 0.4 mg/L, and 0.13 mg/L. 19 oc <: c UJ n. Q oc Q 00 _J <: q: uj I =c UJ a: <: UJ CI SC OC Q. O a; Q- 50 100 150 200 PROPRANOLOL CONCENTRATION yg/L Fig. 8. Propranolol calibration curve from spiked serum extracts Comparison of peak area drug/peak area internal standard with drug concentration in yg/L. Each point represents an average of three determinations. Standards are: 200 yg/L, 67 ug/L, and 22 yg/L. 20 TABLE 2. LINEAR REGRESSION ANALYSIS OF 3 POINT CALIBRATION CURVES FROM SPIKED SERA3 f Correlation Slope Y-intercept Coefficient Procainamide 0.2195 -0.0027 0.9990 N-acetyl procainamide 0.1344 -0.0055 0.9991 kyldisopyramidec Mono-N-deal 0.1531 0.0024 0.9994 Disopyramide0 0.1470 -0.0012 0.9994 Quinidine 0.3426 0.0513 0.9986 Propranolol 0.5435 0.0008 0.9938 Dihydroquinidinee 0.3364 -0.0057 0.9998 Ordinate: Peak area drug or metabolite/Peak area internal standard. Abscissa: Concentration of drug of metabolite standards as indicated below. Standards: 1.3, 4.0, and 12 mg/L. Standards: 0.9, 2.7, and 8.0 mg/L. Standards: 22, 67, and 200 yg/L. e Standards: 0.13, 0.4, and 1.2 mg/L. f raw data used in calculation. 21 TABLE 3. ABSOLUTE EXTRACTION EFFICIENCIES FROM SPIKED SERA AT VARIOUS DRUG CONCENTRATIONS CONCENTRATION: (mg/L) 16 Procainamide 98% 98% 96% 99% 85% N-acetyl procainamide 74 74 73 72 63 Mono-N-dealkyldisopyr amide 99 102 102 108 98 Disopyramide 103 104 103 114 104 Quinidine 100 103 102 112 103 CONCENTRATION: (mq/L) 1.4 0.70 0.35 0.18 0.09 Dihydroquinidine 98% 96% 97 107% 97% CONCENTRATION: (yq/L) 250 125 63 31 Propranolol 73% 70% 76% 72% 22 run variation. Spiked run- serum for the to-run variation study was stored frozen in 3 mL aliquots. The spiked serum was analyzed fifteen times for within-run variation. The run-to-run assays were performed in duplicate, for fifteen days over a four week period. The relative standard deviation for all drugs, in both precision studies, is less than 10% (Tables 4,5, and 6). Data in Table 7 indi cates the variation in retention times over the four week period of the run-to-run precision study. By using the shifts in the internal standard retention time to correct for shifts in the time of elution of the assayed drugs, the reproducability was within 0.1 min over the four week period. This is particularly significant because the microprocessor can then be easily programmed to identify peaks of in terest by the relative retention times of analyte to that of any selec ted standard (9). Chromatograms of spiked sera are presented in Fig. 9. INTERFERENCES Several types of interferences were investigated in the study. Frequently used prescription drugs, over-the-counter drugs, and car diac associated drugs were tested as potential exogenous interfering agents. Lipemic, icteric, and hemolyzed sera were tested for possible endogenous interferences. Heparin and EDTA were investigated as suit able anticoagulants. Dihydroquinidine, a contaminant of quinidine, was identified by thin layer chromatography and quantified by liquid chromatography. Drug Interference The drugs listed in Table 8 were tested for interference with the assay drugs. The drugs were dissolved in mobile phase and dispensed singly into 1.5 mL injection vials, at their respective high therapeutic serum concentrations. The concentration in the vial was made to approx imate the concentration that would result from a hypothetical serum sample that was carried through the extraction procedure. 75 yL of solution from each vial was injected into the chromatograph. The compared the internal standard and resulting retention times were to antiarrhythmic drug retention times. Previous experience with the ef ficiency of the column indicated that drugs eluting within 0.5 min of interfere with the accurate quanti any component to be assayed might fication of that component. Table 9 lists the drugs that were tested 23 TABLE 4. WITHIN-RUN PRECISION DRUG CONCENTRATION mg/L (PROPRANOLOL yg/L) PA NAPA NDAD DISOP. QUIN. DIHYQ. PROPL. 7.3 7.3 3.6 3.3 6.1 0.59 134 7.0 7.1 3.4 3.1 5.8 0.54 124 7.1 6.4 3.7 3.6 6.6 0.64 144 6.5 6.1 3.3 3.1 5.8 0.58 121 7.1 7.3 3.5 3.2 5.9 0.57 131 6.6 5.5 3.6 3.5 6.6 0.64 140 7.2 7.4 3.6 3.3 6.0 0.59 134 6.7 6.1 3.5 3.3 6.2 0.61 132 7.0 6.5 3.5 3.2 5.9 0.59 129 6.8 6.6 3.4 3.1 5.9 0.54 124 6.9 6.8 3.4 3.2 5.9 0.57 125 7.1 6.8 3.5 3.2 6.1 0.58 131 7.0 6.8 3.5 3.2 5.9 0.58 128 7.2 7.4 3.5 3.2 6.0 0.55 127 7.0 6.5 3.5 3.2 6.0 0.55 129 24 TABLE 5. RUN-TO-RUN PRECISION (AVERAGE OF DUPLICATES) DRUG CONCENTRATION mg/L (PROPRANOLOL yg/L) DATE PA NAPA NDAD DISOP. QUIN. DIHYQ. PROPL. 8/15 5.8 5.4 3.9 3.9 6.4 0.60 94 8/19 6.1 6.1 4.0 3.8 6.3 0.59 92 8/20 6.4 6.7 4.0 3.9 6.4 0.60 98 8/22 6.0 6.0 4.0 3.9 6.5 0.60 96 8/24 6.2 6.6 4.2 4.0 6.6 0.73 98 8/26 6.0 6.2 4.2 4.2 7.0 0.76 101 8/27 5.5 5.3 3.8 3.7 6.2 1.19 97 9/4 6.0 5.9 4.2 4.1 6.8 0.75 94 106 9/7 5.7 5.4 4.1 4.2 6.7 0.71 91 9/8 6.3 6.6 4.3 4.2 6.7 0.72 103 9/9 6.0 6.3 4.1 4.0 6.4 0.71 0.77 107 9/10 6.1 6.1 4.2 4.1 6.6 0.69 95 9/11 5.2 4.7 3.8 3.7 5.8 0.69 9/12 5.9 5.9 4.0 3.9 6.2 0.71 94 9/19 5.9 6.2 4.1 4.1 6.4 a Peak area not integrated properly. 25 TABLE 6. REPRODUCIBILITY FOR ANTIARRHYTHMIC DRUG DETERMANATIONS AND REPRESENTATION PEAK AREA RATIOS Within- Run Serum Pool (n=15) Relative Standard Standard Mean Deviation Deviation (mg/L) (mg/L) (%) Procainamide 7.0 0.23 3.4 N-acetyl procainamide 6.7 0.56 8.3 Mono-N-deal kyl disopyramide 3.5 0.10 2.7 Disypyramide 3.3 0.14 4.2 Quinidine 6.1 0.23 3.9 130a 6.1a Propranolol 4.7 Dihydroquinidine 0.58 0.031 5.3 Concentration yg/L. Poo' Run-to-Run Serum 1 (n=15)a Relative Peak Area Standard Standard Ratio, Drug Mean Deviation Deviation Internal (mg/L) (mg/L) (%) Standard Procainamide 5.9 0.30 5.1 1.22 N-acetyl procainamide 6.0 0.55 9.3 0.75 Mono- N-deal kyl d i sopyrami de 4.1 0.15 3.7 0.59 Disopyramide 4.0 0.17 4.3 0.57 Quinidine 6.5 0.29 4.5 2.18 98b 5.0b Propranolol 5.1 0.054 Dihydroquinidine 0.69 0.064 9.3 0.20 a Between assay variation was determined by assaying the pool in dup licate on 15 days over a four week period. Concentration yg/L. 26 TABLE 7. RETENTION TIMES RUN-TO-RUN PRECISION STUDY VALUES OBTAINED OVER A 4 WEEK PERIOD a Rt RANGE (MIN) R RANGE (MIN) CORRECTED Procainamide 5.25-5.53 5.32-5.50 NAPA 7.11 - 7.59 7.32 - 7.49 NPP 10.47. - 10.85 NDAD 14.52 - 14.84 14.68 - 14.78 Disopyramide 17.03 - 17.37 17.18 - 17.31 Quinidine 18.04 - 18.54 18.28 - 18.44 Dihydroquinidine 19.61 - 20.10 19.85 - 20.01 Propranolol 21.30 - 21.82 21.53 - 21.71 a Corrected for shifts in internal standard retention within the run. 27 O1 0.8 UJ o z < CD OC O CO OD < u U W~^ H MINUTES 25 Fig. 9. for serum extracts of Chromatograms (a) blank serum, (b) spiked with drug at comparable detector sensitivity, and (c) serum spiked with drug at reduced sensitivity to illustrate peak shapes and relative heights. The concentrations are equal to those listed in Table 5 for run-to-run precision. The components are: A, procainamide; B, N- acetyl procainamide; C, N-propionyl procainamide; D, mono-N-dealkyldiso- pyramide; F, disopyramide; G, quinidine; H, dihydroquinidine; and I, propranolol . 28 TABLE 8. DRUGS TESTED FOR INTERFERENCE AT THE SERUM LEVELS INDICATED Anticonvulsants Cardiac associated drugs Phenytoin 20 mg/L Methyl Dopa 4.0 mg/L Phenobarbital 60 mg/L Hydralazine 1.0 mg/L Primadone 15 mg/L Chlorthiazide 2.0 mg/L Carbamazepine 10 mg/L Bretylium tosylate 1.0 mg/L Valproic acid 100 mg/L Isosorbide dinitrate 4.0 mg/L Metaprolol 4.0 mg/L Analgesics Furosemide 10 mg/L* Salicylate 400 mg/L Hydrochlorthiazide 4.0 mg/L Meperidine 0.5 mg/L Lidocaine 5.0 mg/L Acetaminophen 40 mg/L Pentazocine 0.3 mg/L Miscellaneous Propoxyphene 0.5 mg/L Theophylline 20 mg/L Quinine 5.0 mg/L Sedative / Hypnotics * Flurazepam 1.0 mg/L No data available on serum le Level calculated on recommend' Glutethimide 4.0 mg/L dose. Methaqualone 5.0 mg/L Meprobamate 20 mg/L Psychotherapeuti cs Diazepam 2.0 mg/L Chlordiazepoxide 10 mg/L Oxazepam 1.0 mg/L Amitriptylene 0.5 mg/L Imipramine 0.5 mg/L Doxepin 0.5 mg/L Chlorpromazine 1.0 mg/L Prochlorperazine 1.0 mg/L Thioridizine 1.0 mg/L Therapeutic concentrations found in References 1,10, and 11. 29 TABLE 9. INTERFERANCE STUDY: DRUG RETENTION TIME AND PEAK AREA Rta Pim Peak Areab mrC Amitriptylene _ NIT Imipramine NR Propoxyphene NR Chlorpromazine NR Thioridizine NR Prochloroperazine 3.2 335,400 Methyl Dopa. 3.3 733,200 Hydralazine* 5.0 7:20,000 (Procainamide) 5.3 1,938,000 Theophylline* 6.0 10,660,000 Acetaminophen 6.3 7,036,000 Bretylium Tosylate* 6.9 149,100 (NAPA) 7.3 1,515,000 Chlorthiazide* 7.3 116,000 Hydrochlorthiazide 8.1 1,921,000 (NPP) 10.6 1,514,000 Salicylate 12.4 10,000,000 Bretylium Tosylate 12.7 219,600 Primadone 14.0 3,162,000 Lidocaine* 14.5 2,920,000 Meprobamate* 14.6 43,030 (NDAD) 14.7 309,300 Metaprolol 16.0 648,000 (Disoypramide) 17.2 956,000 Phenobarbital* 17.3 5,510,000 Furosemide* 18.0 1,583,000 Meperidine* 18.2 88,100 (Quinidine) 18.3 2,680,000 Quinine* 18.4 2,401,000 Pentazocine* 19.5 79,420 Valproic Acid* 19.7 174,000 (Dihydroquinidine) 19.9 196,900 Isosorbide Dinitrate* 19.9 833,600 P-Chlorodisopyramide 20.6 Internal std. Carbamazepine* 20.9 4,440,000 Phenytoin* 21.0 2,799,000 Glutethimide* 21.3 1,767,000 Diazepam* 21.4 223,900 (Propranolol) 21.5 56,240 Flurazepam 22.4 538,900 Methaqualone 22.4 3,071,000 Oxazepam 22.7 626,300 Chlordiazepoxide 22.9 2,719,000 Doxepin 22.9 354,400 a calculated at nm, r -retention time in min. Area by integrator 212 c NR-no response (no detectable elution under assay conditions). * component because of Compound may interfere with an assay its elution characteristics. 30 and their respective retention times. Those components identified with an asterik fulfill the criterion as possible interfering agents. Separate aliquots of pooled drug free sera were individually spiked with the possible interfering drugs at the concentrations indic ated in Table 8. Each was taken through the extraction procedure and injected into the chromatograph. Drug peak areas before and after ex traction were compared to assess the extraction characteristics of each of the drugs. To determine if consumption of quinine water (tonic water) would increase quinidine levels, a volunteer drank 830 mL (28 oz) of Schwepp's tonic water in a 15 min period. Blood was drawn 120 min later for de termination of quinidine by our assay procedure. A review of the drug extraction data from Table 10 shows the fol lowing; an apparent increase in procainamide concentration of 0.2 mg/L by hydralazine and an apparent increase in dihydroquinidine concentra tion of 0.06 mg/L by pentazocine, both of which are not clinically sig nificant. Tonic water consumption would falsely increase quinidine by 0.1 mg/L, also insignificant. Quinine, a stereoisomer of quinidine, significantly interferes with the assay of quinidine. A 5.0 mg/L quinine level would increase quinidine by 5.0 mg/L. Lidocaine, a car dioactive drug with basic properties, will interfere with the quan tification of NDAD. The interference of glutethimide and carbamazepine, neutral ex tracting drugs, with propranolol, was investigated further. Three se parate pooled drug free serum aliquots were spiked with either carba- mazepine (10 mg/L), glutethimide (4.0 mg/L), or propranolol (100 yg/L). An aliquot of each of these sera was processed using the normal assay protocol. A second aliquot of each of the spiked sera was processed through to the aspiration of the organic eluting solvent. Instead of evaporating the HC1 phase immediatedly after solvent aspiration, 8 mL of additional solvent was added to the tubes that contained 200 yL of HC1. The tubes were then vortexed and centrifuged again. The organic solvent was aspirated and then the HC1 phase was evaporated to dryness. A third aliquot of spiked sera was taked through two additional solvent addition/aspiration steps. After the first 8 mL of new extraction sol vent had been aspirated, another 8 mL was added. These tubes were then vortexed, spun, aspirated, and dried. During the aspiration step, 31 TABLE 10. EXTRACTION DATA ON COMPOUNDS WHICH MAY INTERFERE BECAUSE OF THEIR CLOSE ELUTION TO AN ASSAY DRUG Serum cone. Peak Area Peak Area Druq (mq/L) Pre-extract Post-extract % Recovery Chlorthiazide 2.0 119,200 1,100 0.9 Meperidine 0.5 521,900 NRa 0 Phenytoin 20 3,040,000 NR 0 Furosemide 10 3,610,000 NR 0 Meprobamate 20 40,200 NR 0 Diazepam 2.0 251,500 NR 0 Valproic acid 100 160,000 NR 0 Bretylium 1.0 120,600 NR 0 Isosorbide 1.0 178,000 MR 0 di nitrate Theophylline 20 12,835,000 NR 0 Phenobarbital 60 6,830,000 NR 0 Hydralazine 1.0 660,000 193,950 29.4 Lidocaine 5.0 2,739,000 2,503,000 91.4 Quinine 5.0 3,195,000 3,286,990 102.9 Carbamazepine 10 4,706,000 555,700 11.8 Glutethimide 4.0 1,944,000 193,060 9.9 Pentazocine 0.3 72,640 36,050 49.6 - - Tonic water _ 400,800 a NR-no response, no detectable elution under assay conditions. b Area calculated by integrator at 212 nm. 32 approximately 7.5 mL of the 8 ml of organic solvent can be removed without disturbing the 200 yL HC1 layer. In this way the initial ex traction and back extraction steps remove 94% of neutral extracting compounds. A second solvent addition step increases the removal of these interfering drugs to 99.6%. A third addition would theoretically remove 99.98% of all neutral extracting compounds. The results of this experiment are tabulated in Table 11. Both carbamazepine and glutethimide can be eliminated by the extra extraction steps. However, with each successive step there is a 30% loss of propranolol. After the third extraction, a 100 yg/L serum propranolol has a peak area of 25,000. This could present a detection problem with intermediate propranolol levels. There is no apparent loss of internal standard in these extraction steps. The 82% recovery of internal standard is therefore probably due to a loss of 18% in the column. A propranolol serum standard should therefore be taken through the multiple extraction steps along with the patient's specimen to com pensate for extraction losses. Endogenous Interference Lipemic, icteric, and hemolyzed sera were tested for possible in terferences with the assay drugs. Four pools were prepared of normal, lipemic, icteric, and hemolyzed sera. Aliquots of each were spiked with the assay drugs at concentrations to represent both high and low ther apeutic serum levels: High Low Quinidine (mg/L) 5 2 Procainamide (mg/L) 5 2 NAPA (mg/L) 5 2 Disopyramide (mg/L) 4 2 NDAD (mg/L) 2 0.5 Propranolol (yg/L) 100 30 DIHYQ (mg/L) 0.6 0.2 Each specimen was then extracted in duplicate.cate. Equivalent serum blanks were also run as controls. All sera, regardless of whether or not a potential endogenous in terference was present, had elution peaks at 4.57, 9.02, and 19.27 min. These substances, do not coelute with any of the assay drugs. Lipemic sera also had an endogenous peak at 12.34 min, area 175,000, that did min not interfere with the assay. Another peak, that eluted 0.17 after 33 TABLE 11. DATA ON THE ADDITIONAL EXTRACTION STEPS WHICH ARE REQUIRED TO REMOVE THE INTERFERING DRUGS CARBAMAZEPINE AND GLUTETHIMIDE FROM THE PROPRANOLOL ASSAY. Apparent Propranolol Level (yg/L) Extractions Carbamazepine9 Glutethimide9 Propranolol' Pre-extraction 5129 2693 135 Extraction Protocol 733 178 99 One Added Extraction 39 0 73 Two Added Extractions 0 0 45 Individual serum aliquots were spiked at the following concentrations; carbamazepine (10 mg/L), glutethimide (4 mg/L), and propranolol (100 yg/L). Drug standards were prepared in mobile phase at the indicated con centrations and chromatographed without extraction. 34 produced the NDAD, following areas in extractions of drug free sera: hemolyzed (22,100), lipemic (222,400), icteric (52,700). The extract of normal serum did not have this peak. A NDAD serum level of 0.5 mg/L produces a peak area of 170,000. Lipemic and icteric sera would sig nificantly increase NDAD concentrations (Table 12) and should therefore not be used for analysis of this metabolite of disopyramide. Results of spiked sera (Table 12) show an elevation of NDAD levels in both li pemic and icteric specimens. Other results, such as the apparent de crease in propranolol in the low therapeutic spike of hemolyzed serum, could not be explained on the basis of the drug free serum data. Ic teric sera also show an apparent procainamide increase. No explanation is offered for these data. Because of the problems related to the use of lipemic, icteric, and hemolyzed sera, they should not be accepted as appropriate samples for analysis of the antiarrhythmic drugs. EDTA plasma and heparinized plasma were tested for suitability in assaying the antiarrhythmic drugs. Seventy ml of whole blood, one heparinized tube and one tube containing EDTA were drawn from a volunteer. Approximately sixty ml of the whole blood sample was quickly added to five ml of normal saline that had been spiked with drugs. The blood was mixed and transferred to tubes that contained either EDTA, heparin, or no anticoagulant. All serum and plasma samples were removed from the cells within one hour. The concentrations of spiked drugs were approximately as follows: quinidine 5 mg/L, procainamide 5 mg/L, NAPA 5 mg/L, disopyramide 4 mg/L, NDAD 1 mg/L, dihydroquinidine 0.4 mg/L, and propranolol 100 yg/L. Each of the three specimen types was extracted in duplicate. Blanks of each specimen type were also analyzed to determine if any anticoagulant related interferences are present. No interfering peaks were seen upon extraction of the blank serum or either of the plasma. It appears from the data in Table 13 that EDTA plasma, heparinized plasma, or serum may be used for all drugs except NAPA. EDTA plasma increases NAPA values and should not be used for metabolite. The reason for this increase assay of the procainamide of could not be explained on the basis of the results the drug free extracts. No explantion is offered for this increase. Dihydroquinidine was identified as a contaminant of the quinidine sulphate preparation in two ways. First, the retention times of a known 35 a,b TABLE 12. ENDOGENOUS INTERFERENCES LOW CONCENTRATION SERUM TYPE PA NAPA NDAD DISOP. QUIN. DIHYQ. PROPL. Normal 1.87 1.74 0.59 2.55 2.84 0.26 33.9 Hemolyzed 2.15 2.49 0.64 2.33 2.49 0.22 22.6 Icteric 2.29 2.06 0.74 2.62 2.92 0.23 30.6 Lipemic 2.00 1.71 1.38 2.60 3.15 0.25 29.5 HIGH CONCENTRATION SERUM TYPE PA NAPA NDAD DISOP. QUIN. DIHYQ. PROPL. Normal 4.91 4.55 2.47 4.89 6.82 0.65 130 Hemolyzed 4.87 5.43 2.39 4.44 6.09 0.56 105 Icteric 5.54 4.89 2.88 5.49 7.65 0.68 124 Lipemic 5.04 5.12 3.16 4.57 6.69 0.60 104 Apparent drug concentration (mg/L), propranolol (yg/L) Average value of duplicate samples. 36 TABLE 13. SERUM VS PLASMA SUITABILITYa'b PA NAPA NDAD DISOP QUIN DIHYQ PROPL EDTA 5.00 7.84 0.99 5.37 5.51 0.49 72.5 Heparin 4.80 7.04 0.98 5.59 5.44 0.48 70.2 Serum 4.79 7.01 0.98 5.33 5.50 0.48 74.5 a Apparent drug concentration (mg/L), propranolol (yg/L). Average value of duplicate analyses. 37 solution of dihydroquinidine and the contaminant peak matched exactly. Secondly, 20 yg each of quinidine (and the contaminant), dihydroquini and quinine (a dine, stereoisomer of quinidine) were spotted separately on two silica plates for thin layer chromatography. The Rf's of the contaminant and dihydroquinidine, with two different mobile phase com positions, agreed within experimental error (Table 14). All substances were detected by both short wavelength UV fluorescence and iodoplatinate spray. CORRELATION Procainamide, NAPA, quinidine, and disopyramide were assayed in patient's sera by our liquid chromatographic method and compared to commercially available enzymeimmunologic techniques (EMIT) (13). Sera for determination of procainamide, NAPA, and quinidine were collected and stored frozen at C until the. time of assay when each specimen was thawed and vortexed for 5 s to insure homogeneity. Pooled drug free sera was spiked with disopyramide at various concentrations. Each sample was assayed once by our liquid chromatographic method and in duplicate by enzymeimmunoassay. Results by enzymeimmunoassay that exceeded the method linearity for quinidine and disopyramide (both 8.0 mg/L), or for procainamide and NAPA (both 16.0 mg/L), were diluted with drug free serum and reassayed. Dihydroquinidine was added to drug free serum at concentrations of 0.5 mg/L and 1.0 mg/L and assayed in triplicate by EMIT. Dihydroquinidine is stated in the Syva brochure to increase a quinidine value of 2.0 mg/L by 30% when present at a concentration of 0.6 mg/L (i.e. both quinidine and dihydroquinidine are equally effective antigens in the technique). Quinidine Correlation Assay of dihydroquinidine spiked sera by EMIT gave the following results: Dihydroquinidine Added Quinidine Found 1.0 mg/L 1-0 mg/L (average) 0.5 mg/L 0.5 mg/L (average) Dihydroquinidine exhibits 100% cross-reactivity with the EMIT assay and the antibody for quinidine. Because of this cross-reactivity method to resolve the two ability of the liquid chromatographic drugs, the correlation was performed using quinidine (HPLC) vs quinidine 38 TABLE 14. THIN LAYER CHROMATOGRAPHY OF QUINIDINE (AND CONTAMINANT), DIHYDROQUINIDINE, AND QUININE fb Phase3 Mobile Quinidine (Contaminant) Dihydroquinidine Quinine 68:8:4 0.50 (0.44) 0.43 0.41 68:8:8 0 .57 (0.51) 0.52 0.49 a Volume of ethyl acetate:methanol :NH.0H (cone). b Compared to mobile phase migration of 13.5 cm. Detection by short wavelength UV fluorescence after H2S04:abs. ethanol (90:10) spray and also by iodoplatinate spray (Reference 12). 39 (EMIT), and quinidine plus dihydroquinidine (HPLC) vs quinidine (EMIT). The correlation data are found in Table 15 and Figure 10. The correla tion is only slightly affected by the inclusion of dihydroquinidine in the calculations. Least square regression analysis of the two sets of data: Quinidine + Dihydroquinidine (HPLC) vs Quinidine (EMIT) = = r .9508 HPLC 1.11 (EMIT) - 0.13 n = 25 Quinidine (HPLC) vs Quinidine (EMIT) - = - = r .9554 HPLC 1.08 (EMIT) 0.14 n 26 This is probably due to the low levels of dihydroquinidine in serum. The liquid chromatographic assay for quinidine correlates well with enzyme immunoassay technique. The correlation coefficient, slope, and Y-inter- cept are all acceptable for clinical use. At present, there is insuf ficient data on the clinical significance of dihydroquinidine (14,15). We are reporting quinidine values and keeping records of both compounds for possible future use. Procainamide / NAPA The correlation between HPLC and EMIT for procainamide and NAPA, as well as the least square regression analysis of the data, are found in Figures 11 and 12, and Table 16. The ratio of NAPA to procainamide may reflect the metabolism of procainamide. In patients on chronic procainamide therapy, high ratios indicate fast acetylation while, low ratios indicate slow acetylation. Acetylation is genetically determined (1). Very high ratios also may indicate renal insuf ficiency due to the accumulation of NAPA. NAPA has a half life of 6 hours compared to 3 hours for procainamide (1). Disopyramide / NDAD Because of the infrequent use of disopyramide compared to the other antiarrhythmics, patient specimens were difficult to obtain. The method correlation was therefore performed on pooled drug free sera which had been spiked with disopyramide at several concentrations. The correlation between HPLC and EMIT for disopyramide, as well as the least square regression analysis of the data are found in Figure 13 and Table 17. There is no readily available comparative method for the analysis of NDAD. Quantification of NDAD may prove valuable because of its reported high cholergenic potency (16). Reported serum levels of 40 TABLE 15. COMPARISON OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH ENZYMEIMMUNOASSAY FOR THE DETERMINATION OF QUINIDINE (mq/L)b HPLC EMIT + Quin Dihyq . (mq/L)a Total0 Specimen Dihyq Quin Dihyq % 2643 2.5 2.8 2.6 .16 5.8 6643 0.6 0.6 0.6 .01 2.0 8394 3.3 3.3 3.3 .05 1.5 3243 3.4 3.1 3.0 .07 2.3 2449 3.1 3.5 3.3 .20 5.7 8793 2.5 2.2 2.2 .03 1.3 6527 0.4 0.3 0.3 .02 6.3 3252 1.5 1.4 1.4 .04 2.8 2885 2.9 3.4 3.3 .14 4.1 5454 2.2 2.1 2.0 .09 4.3 4251 3.2 3.7 3.6 .10 2.7 105 1.7 0.7 0.7 .04 1.7 2976 4.0 4.5 4.4 .10 2.6 1607 5.0 5.5 5.3 .18 3.3 3042 2.8 3.1 2.9 .21 6.7 2251 2.4 2.5 2.3 .22 8.7 4622 2.1 2.0 1.9 .05 2.6 3753 2.3 2.0 2.0 c - 2843 1.9 2.1 2.0 .08 3.8 4439 2.5 2.6 2.5 .06 2.3 3986 1.8 2.7 2.7 .04 1.5 2710 1.4 1.4 1.3 .12 8.5 9264 1.2 1.5 1.4 .08 5.4 4010 0.9 1.1 1.0 .07 6.5 5438 3.2 3.3 3.2 .08 2.4 4220 2.6 3.2 3.1 .09 2.8 Average of duplicate assays. EMIT crossreacts 100% with dihydroquin idine (see text). EMIT result is therefore a sum of quinidine and dihydroquinidine. HPLC is capable of quantitating both quinidine and dihydroquinidine. Dihydroquinidine levels shown to illustrate its low concentration in patient serum. Peak area not integrated properly, therefore not included in the correlation Dihyq % Total =( Dihyq/ (Dihyq + Quin)) x 100. 41 - 1 - :-::- Ef x ". Xi -HIfP :. : ElH. ' -A- ' XE - - - - 1 EfEXXXEIj Ei - .!... '-'- ZZ.. iZZ- . ;. !'! - - iEE Ef XT. - ._r|_ IEEE frljXf .. E ! if ! Z.zzLzA: :.._ . . 1 fXtXE En. f : ZZZZZ i -yy\y~- - E- d.E!i " .H!. - -";:--.e. AAXE - E i f-EXE::: AAZZ AzzzAZZ ------. EXPE .-jf 7TPii --; X- - - iil 1 ..-_ 11 .::._ - X.EE: 7 :~e' nX - Ei i EEElj . -e fiEiifi Elie: EilnT .ZZ pE.i EE -: p- '-" ^ ... EXEe - : -t -ee i 1 ::ii _. 1 :[Efpi E ii|!H. _ ._. _ i J ;_. _. _ ' !.-' . EiE : e ". ': z ' "T ZZZZZ ' - TTEfTX _f I- E - 1 - .E-fi.iilEii|liEeXee ZZZZ EE|-..E 4e? i- - -1 ' - - - 1- "- i . idi . -r Xid zzzz zzzz I . f "~X . -.Lie EiEEfE EE "EP EfEE z-'-^r- ^ 1 T_, EEEf"H!E" 1 ee ...... _ . ' HHH j 1 -EHE 1 - E. .EE .__..:E-U-i-. _^ ------. EEIHli nil EX Ei. XPnlf - - i r . .. ------._ x- 1 A- - " EEffE" + '.ZZZZ'' XIX... . EE:..Ei EElii! ZLZ' - iEEjli.l !E! 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ET" . E. i XE XX - - y Z'S.ZZf ,. -J :-:h; eie!e -.:di.. " - Xf Q- ffiiEE E OEH EEHI"., 1 zz Ij zz Erff: 'r'~r-.:- i-.E-: - riii/.i:i-ii. .1 iiHilE. EH : */ xx-_: . -e rjni . . ^ - I o X-zAzz^Zzz-^ d IXlj - -- - - 1-- - . - i J n : 1 r r y \ ! j ---! Ol -- ... 4- . . - . Hi - - - - .. . -1-.. .. M|-E- 1 1 -1 -, XXEEi-EXX IH H \ "^ j E _ y~\ - X 1 - lE'f.f .1 - Ep" UJ IiEpX ii! ... ZZ : EH E. EEeE! " ' """" -- ~-l "1 - - ; I I :: i - Xf X--- z'z EpE- - P-"- -EXIf.I t d - -- -v- P- !Ej:::! xeeixexx: E.EEEi I I EfXE XX IP X / 1 h , -z h1 X XE ! Epf I XTrEE : IE PE :i : "i. _ : ElpHi ~" - r*r'\\*.-.m o / "' o- ' T--fE" - r-i- EE ! j -ii-i-i-E1-"-- .. A,-. CM EEEfE i .: yz 'EEjXf 1 EEXL EE1"! - 1 ii 4:--- 1: : HiiEni i d : ""-_ -XE / i XEXT^ a-iOir * ..n.^tb. J --:- ._. ..z,.zz.z E4 1 eeee Yzzzz^-: "-zfr X - " fviXftJxsCtvFJE.dE X V ~'\ / - ; ZZZZZ"X":. _E HX XEEX XEEE4-rE0p^9 JOl^E- .XEXE y~ H E.T 1 ^- " o i_E i - 1 . | 1 r-i - hiTTXA EEEEH . . . - - -X i-d- Azz'czzzzz EXEEEEXE , i Ei.#ti-i EX-T- ZZZZ." EX-"-t. ix/iii EE " ' f "xl X r ". \ r \ ! 1 -j- t- -4 - ^ _ ! 1 X ie h ----- tn .. - - - E t i- /*- H .!! HIE . : ill X x~- 1 iz zz_ 1- "" X n 1.0 2.0 3.0 4.0 5.0 6.0 QUINIDINE mg/L EMIT METHOD Fig. 10. Comparison of the high performance liquid chromatograph method (X-axis). for quinidine (Y-axis) with enzymeimmunoassay 42 TABLE 16. COMPARISON OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH ENZYMEIMMUNOASSAY FOR DETERMENATION OF PROCAINAMIDE AND N-ACETYLPROCAINAMIDE Procainamide (mg/L) NAPA (mg/L) NAPA & b EMIT3 EMITa Sample # HPLC HPLC NAPA/ PA Total A-l 14.5 15.8 d d - d A-2 13.6 14.5 16.5 15.3 1.1 29.8 A-3 4.7 4.7 6.7 6.8 l.-\ 11.5 A-4 1.3 1.3 1.8 1.8 1.4 3.1 A-5 2.9 2.3 3.5 2.9 1.3 5.2 A-6 7.1 6.2 d d - d 4.7 A-7 1.0 0.9 3.9 3.8 4.2 4.8 A-8 2.5 2.5 2.5 2.3 0.9 6.2 A-9 2.5 2.3 5.4 3.9 1.7 - d B-l 6.0 4.4 d d 6.3 B-2 2.8 2.5 5.0 3.8 1.5 0.8 6.7 B-3 4.2 3.8 3.8 2.9 1.2 2.2 B-4 1.3 1.0 1.8 1.2 1.0 11.5 B-5 5.8 5.7 6.7 5.8 6.4 B-6 3.8 4.0 2.6 2.4 0.6 1.1 H.9 B-7 5.9 5.8 6.4 6.1 3.9 0.7 9.5 B-8 6.3 5.6 5.0 0.8 0.7 2.0 B-9 1.6 1.2 1.3 0.8 1.6 1.3 C-l 0.5 0.5 1.0 1.5 0.6 4.0 C-2 2.8 2.5 2.3 3.5 0.7 8.5 C-3 5.3 5.0 5.5 d - d C-4 8.3 8.3 d 10.4 11.0 0.9 23.5 C_5 10.3 12.5 1.7 0.5 5.3 C-6 3.4 3,6 2.5 6.4 5.4 0.4 20.1 C-7 14.0 14.7 4.1 1.1 7.9 C-8 4.3 3.8 5.0 8.9 8.1 1.2 14.7 C-9 6.2 6.6 a Averaae of duplicate assays. , Reference *> rate of ation (see 1) UsefSl for determining the acety well as the sum of c management procainamide as For o t mum patient Reference 1 . should be reported (see procainamide and NAPA, correlation not included in the d method linearity, Values exceeded the tested 43 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 PROCAINAMIDE mg/L EMIT METHOD Fig. 11. Comparison of the high perfromance liquid chromatographic method for procainamide (Y-axis) with enzymeimmunoassay (X-axis). 44 12.0 14.0 2.0 4.o 6.0 8.0 10.0 16.0 N-ACETYLPROCAINAMIDE mg/L EMIT METHOD Fig. 12. Comparison of the high perfromance liquid chromatographic method for N-acetyl procainamide (Y-axis) with enzymeimmunoassay (X-axis). 45 TABLE 17. COMPARISON OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH ENZYMEIMMUNOASSAY FOR THE DETERMINATION OF DISOPYRAMIDE Specimen9 EMITb HPLC A-l 0.5 0.6 A- 2 1.0 1.0 A- 3 1.6 1.5 A-4 2.2 2.3 A- 5 2.1 2.3 A-6 2.7 2.9 A- 7 2.6 2.2 A-8 3.3 3.2 A- 9 3.8 3.3 B-l 4.0 3.6 B-2 4.4 4.2 B-3 4.5 4.5 B-4 5.9 4.7 B-5 4.8 4.5 B-6 5.8 5.3 B-7 5.8 6.5 B-8 6.8 7.5 B-9 6.7 6.1 C-l 7.7 7.9 8.6C C-2 10.6 9.7C C-3 9.8 4C C-4 10. 11.5 0C C-5 13. 13.1 a Drug free serum was spiked with disopyramide at various concentrations. Average of duplicate assays. Values originally exceeded method linearity. 46 ra o o _i a. cc >- o 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 DISOPYRAMIDE mg/L EMIT METHOD performance liquid chromatographic method Fig 13. Comparison of the high for disopyramide (Y-axis) with enzymeimmunoassay (X-axis). 47 NDAD are within the linearity range of our assay, but the exact antiar rhythmic activity of this metabolite is uncertain (1,17). We are cur rently reporting disopyramide concentrations and measuring NDAD levels for possible future use. Propranolol At present, insufficient numbers of patient samples prevent us from acquiring correlation data for propranolol. The liquid chromatographic method for analysis of propranolol will be compared to our fluorometric method (4). TIME COST STUDY The time required to process individual runs of 4 specimens and 20 specimens was determined (Table 18). The total cost involved in the analysis of one specimen, when three specimens and one control are assayed, was also determined. These data are presented in Table 19. There is a substantial savings in technologist time with the liquid chromatographic procedure compared to the methods formerly used. Assay of four specimens for propranolol by fluorometry requires 2.0 hours of technologist time (4). To assay the same number of patient sera for procainamide by spectrophotometry, 1.5 hours of technologist time is required (5). The total cost, however, is higher with liquid chromato graphic analysis than with the manual methods. This is due primarily to instrument overhead. Enzymeimmunoassay, which is a precise and specific technique, is also an expensive method for analysis of these drugs. The total cost of performing an EMIT assay for either quinidine, procainamide, NAPA, or disopyramide is $5.60 for a run of 4 patients Liquid (based on methodology applied to the Abbott ABA-100) (13). expensive than EMIT when more than chromatography is therefore less one drug is requested for assay on a patient's serum. 48 TABLE 18. TIME REQUIRED TO PROCESS THE NUMBER OF SPECIMENS INDICATED THROUGH THE ENTIRE ASSAY PROCEDURE Process 4 Specimens Process 20 Specimens Techa Stepb Techa Stepb Step Time Time Total Time Time Total 1. Setup. Assemble all -Mil) (min) (min) (min) (min) (min) materials, dispense buffer, 12 12 12 35 35 35 serum, and solvent twice. 2. Add third solvent, allow 1 7 19 17 52 columns to drain. 3. Vortex tubes (back- 2 2 21 12 12 64 extraction). 4. Spin, aspirate solvent. 4 15 36 15 30 94 5. Evaporate, add mobile phase, and transfer to 5 20 56 20 30 124 injection vial . 6. Run on chromatograph 12 120 176 12 600 724 7. Calculate and report 2 2 178 10 10 734 results. Totals: 38 min 3 hr 111 min 12.2 hr Represents time required for technologist to perform task. Represents time to complete task. Represents time from beginning of procedure. 49 TABLE 19. TOTAL COST INVOLVED IN THE ANALYSIS OF ONE SPECIMEN WHEN THREE SPECIMENS AND ONE CONTROL ARE ASSAYED. Summation9 Cost Amount Technologist time $1.74 a $11.00/hr) $0.75c Detector lamp use $0.55d Analytical column use Elution solvent $0.18 Extraction solvent $0.08 Clin-Elute column $0.80 $0.40e Disposables $5.16f Instrument Total $9.66 Includes the cost of assaying the control serum . Includes wages and benefits. (Lamp cost / Life time in hr) x hr / run. Column cost / number of injections. e Paper, vial caps, etc. f cost. Based on 10 year depreciation and service contract 50 METHOD DEVELOPMENT The development of the HPLC antiarrhythmic drug assay consisted of initially investigating the chromatographic conditions that would affect drug elution from the analytical column. The instrument parameters were then studied in an attempt to optimize the assay for maximum sensitivity. The chosen system was then used for quantitation of the assay components. Several organic solvents and procedures were also tested for their ability to extract the antiarrhythmic drugs from spiked serum. MOBILE PHASE DEVELOPMENT Most of the experimental work involved in this study was directly related to the development and optimization of the mobile phase. The mobile phase conditions that were sought would separate and elute all components of interest in a reasonable length of time. Several par ameters were investigated: Composition (percentages of methanol, aceto nitrile, phosphate), pH, and phosphate concentration. Both isocratic and gradient systems were used to elute the drugs. Flow rate and column temperature were also investigated. Working solutions of drug standards were prepared in a similar manner for all experiments. Methanol stock solutions of each drug were grouped into three vials. Drugs with similar retentions were put in separate groups to help prevent misidentification. Concentrations of 30 - 50 mg/L for each component were used to approach full scale recorder deflection at a setting of 0.768 absorbance units. full scale (AUFS). The solvent composition of the vial always corresponded to the starting com each of the experiments position of the mobile phase being evaluated. In varied while the one of the following chromatographic conditions was values: flowrate 1.0 others were held constant at the indicated ml/ min., pH 3.5. temperature ambient, phosphate buffer 25 mmol/L Mobile Phase Composition The percentage composition of the organic component of the mobile changed phase was varied. The percent methanol :acetonitrile was from 100:0 to 0:100 in several steps. The ratios of methanol :acetonitrile that were used were 100:0, 80:20, 70:30, 65:35, 60:40, 55:45, 50:50, 40:60, 30:70, 20:80, and 0:100. 51 LU OC 0:100 20:80 40:60 60:40 100:0 RATIO METHANOL : ACETONITRILE methanol,' Fig. 14. Effect of the acetonitrile ratio of the mobile phase on drug retention. Drugs were eluted using gradient analysis in which the ratio of organic (methanol.acetonitrile)/phosphate changed from 20:80 at 2.0 min to 70:30 at 20 min. Components are: A, procainamide; B, NAPA; C, NPP; D, lidoc; E, NDAD; F, 4-0H prop!; G, disop; H, quin; H-ihv/n- T anrl .1 '""'"Opl 52 As shown in Figure 14 the retention times of all assay drugs increase directly with methanol concentration and, conversely, they decrease with increasing acetonitrile concentration. The change in retention time is var iable from drug to drug, and results in changes in the elution order of some components. At methanol concentrations of 0 - 40% quinidine is not completely resolved from disopyramide. At 100% methanol, dihydroquinidine is not completely resolved from propranolol. For most assay drugs resolution improves as methanol concentration is increased from 40% - 80%. The early peaks broaden and tail severely with high methanol con centrations ( 80 - 100%). The baseline shows an upward drift with time due to an increase in the absorbance that results from an increase in the gradient methanol concentration. At 100% methanol, the increase in base line absorbance represents a 20% full scale deflection at the end of the run (instrument attenuation set at 0.768 AUFS). Runs with ace tonitrile concentrations greater than 60% have significantly smaller baseline increases. The baseline differences, although noticeable, are not analytically significant because they occur gradually, and thus do not affect the instrument's ability to properly integrate all of the peaks. The slight baseline increase occurring late in the run is due to increasing methanol in the mobile phase which is a function of the gradient design (see Experimental section, Chromatographic Conditions). Figures 15, 16, and 17 illustrate differences in the elution and baseline of six assay components at 100% methanol, at 100% acetonitrile, and at 60/40 methanol/acetonitrile, which was the ratio adopted for the assay. This ratio was chosen because all drugs were well resolved. Phsophate Buffer pH The effect of the buffer pH on the chromatographic elution of each drug was studied. Analyses were performed at pHs of 3.5, 4.5, 5.5, and 6.5. In general, an increase in pH increases drug retention time (Figure 18). This is expected because all drugs in the study have basic properties: = Lidocaine pK = 7.85 Quinidine pK 4.0,8.6 = Disopyramide pK = 8.34 Procainamide pK 9.2 Propranolol = 9.45 (The pKs of NAPA, NDAD, and dihydroquinidine were not available.) 53 IN. STRPT 8. 74 1 1.92 J 15.40 17. 93 i s%ft-.3ft 21 24.9" :E t-p 1080 B Fig. 15. Chromatogram of several assay components which were separated using a mobile phase organic ratio of 100% methanol: 0% acetonitrile. The components with their respective retention times (min) are: pro cainamide (6.07); NAPA (8.74); NPP (11.92); lidoc (15.40); disop (17.99); and dihyq (21.21). 54 I H J ;.thrt ~3 : i-B 3 4:fi IB. 39 D 11 iWAc 14. 14. ? il . 68 \ 234. Fig. 16. Chromatogram of several assay components which were separated organic ratio of 0% 100% acetonitrile. using a mobile phase methanol: are: procain The components with their respective retention times (min) lidoc amide (4.71); NAPA (4.91); NPP (7.20); (11.19); disop (14.27); and dihyq (14.89). 55 STfiF' In.i 5. 34 7 . 04 IB. Z3 14.34 c => 17.18 ~^\%:*rt -*fc- 1 9.71 S4- Fig. 17. Chromatogram of several assay components which were separated organic ratio of 60% methanol: 40% using a mobile phase acetonitrile, with their respective re (assay protocol conditions). The components tention times (min) are: procainamide (5.34); NAPA (7.40); NPP (10.56); lidoc (14.34); disop (17.18); and dihyq (19.71). 56 1 .:. . _ . --- :.j t- -- - : zzzz -- j- TnE - " ET Tr !Hi XE - - : T ; t -m - I - ... . f- "r E . j". A z-zizz:\ - f- .! EX xxfff ii! fi TEH! 'ZZ r- _t- 1 " " - - . i - . "- - - H .ii n : Hi . _ if i -0 i! .. ^ "e f T : nn 1_ - X .. . ._ ==~ i.X.i ... _ . EE !H!i_ - f -Ei-.ff . .. -E E XX 0 ii TTTXr.X ._.j r - -- -i -|nn- i ^=Lz.!.!!!- ~ ~ -f-r :.~ -r- " -j, ii XE ,- . HiXf zzz x '^ V _j I _xx4___t: - " - - " |H zzxz ZZ -. -"---] "1 -4- . -X .:...... zzzZzz- - Tl . _ P~X J. (. J 1_. XT" xhi EX". 'TT - - - zzzZzzA. the; . r 1 _ _- f - ii- ZZZ -E-f|Tin- i.iiX---n iH .. ::EX:: i':X ----- e '1-X:-Iz ex EX zzz.. EX-EX XTfpETxx:xx J CO -i - ; *J" ~"' CM ! . . H H -Hi.ZZZA. ZZ zzzz. i __r_. .Hi! ... " " -H-X ' _., -ffdE-f - . z~z . _. : 1 r [ f J ^^__ i , -- 1 ._4 i - r~ - ^ - : XE Z -..iij-.Hi -:"t-H_-| ETX iXEEffi '_--. :.-. 'zAy/L -E-hh. - , -- ' Eip:ix- -- ii-H iil.. t ^ -- I . - b>^x"" 1 . . - "1 tEiH Z~ - !- Ei >f E TXX-X XEpX -y^ - - =~H I EX "X^iftiXT -- H'iH-fiiH- CO ~Hi iii!- - Ei ^Xh^- "f -i -^diiij EXX:.- -,-"------i --- zzz ; _.._ 4 ! -J n -i ii | y*zz^_ ~z zytTx"Hi! . -f nnnn.n - . EEE-.n- 1 ZZ -H. . 1 Hi!xxSe - .; X-Hi "zz-zzr-z-z. -Ei. - i*/'i?''-|!i/ *- E_- E-J - 4 -- zzzz. n-.i:.ii.i]i.ii:..H..i ~ X . . t . . ' ' X" ^-.-. -:- \ XE1*- - px j !'i-iHi.:EnH zzzz'zi iiiEii-- p ;r_._-X .... ._ -J -. - 1_- 1- ' ?^ - ZZZZ F-: ^^^ZA H riHH i --ZZZ. -, X. ii - E teLJ -^"m f i -E" :i" -L------. iHiii^'Ei-ii; ^EZ< -XdX i i Hi" - _ : a zzzzzzzzzzz I , -. i -iH . fx-.fx4-:x ^ _ EE-Xf! 1 UJ r" ! " ' J* r s. 7Z. r-l ! : \zz~zz EETX^EX - ,_.. -t-..r--TiEJEiT LU ;- -< f EH Z"zz j 1 . i i 1 " LU 4 ^-Hi- .! ii.HH-1 OC 1 >- XffEfX HHit; ~. H XEX-EI XXX d-t.Ef] fX[EX -^ !--.__ 1 r ,....|....n "- " T" -i EETXTJEff XE EH-| _-.HJ.Hi_-J - - + ' -p - . . -- - .__._^>-tE f eL xx!iii^i_i!].i-H 7i! :. zX-z } ! -: HHitiiHi ; ._ -. - v in n ._:_: 1. 1 I -- _L _ _ T xtx-hhXXE'-- -- XE X :fffX_4?-ii^XE l = i I . ,.. . rE;^ Z - ~r~-.'-- }X_ 1 - n. .__:_ l[ 4 nXXiXnETnE; . > j : 1__. ---- 1 j j . ! ] ... . ,. .. __. E; 1 HiiH ..... eex1 j -HH.Hiii . ' __i.ninHiiiHHJ!HH. ZZZZZ ZZZZZ V=H *"B u i. - i E-- XfEErE -- _--! nX Eil .-T^ ---_Z3 CO - _...^ : - -i--- - 1 j^iii _, 4 1 4 ^-Ei-EE^ - Ae ^_H_J^ S ~ i ' '_rz . X T E Eh^X "^" 5iX - TEXXXEETX. Hi. Hi !"! ----- Eiiii XeJ . v\ . !_ H : |- -__!!!.._! -- - -l - -ii E - - _E= 1 ~-E r J 1 1_ q E-JX^j x- - txtEX .. . L . . - -t-~- . iijHH-n <=* - - E..E: Tf f .- -1 e- - -- Hi!, i - ~ i i . -x XX ^ j - i . . . i.iii 1 3.5 4.5 5.5 6.5 PHOSPHATE BUFFER pH Fig. 18. Effect of phosphate buffer pH on drug retention. Components are: A, procainamide; B, NAPA; C, NPP; D, NDAD; E, 4-OH propl ; F, disop; G, propl; H, quin; I, lidoc; and J, dihyq. 57 pH these drugs are in an At low ionized, hydrophilic form and they would be expected to elute quickly from the nonpolar, hydrophobic phase. At high pH stationary they are unionized and, hence, are retarded on the column. The large increase in the retention time of lidocaine is a probably result of the drug's relatively low pK and it's subsequent decrease in ionization at pHs 5.5 and 6.5. At the selected pH for the assay (3.5), all drugs, except NDAD and lidocaine, are sep arated at least one by min. As the pH increases, propranolol converges on dihydroquinidine. Further pH increases result in the propranolol converging on quinidine. Only at pH 6.5 are they again well separated. At pH 6.5, lidocaine is well resolved from NDAD. If lidocaine were to be included in the assay, this would be the appropriate pH. Phosphate Concentration The concentration of the phosphate buffer was varied over the concentration range of 12.5 mmol/L to 100 mmol/L. The optimum amount of phosphate would be the lowest concentration which would allow good resolution in a reasonable time, and still maintain an adequate buf fering capacity. High phosphate concentrations may precipitate in the capillary lines of the liquid chromatograph (internal diameter of 0.0001 inch). A slight increase in retention time for each of the drugs, is seen as the concentration is decreased from 100 to 25 mmol/L (Figure 19). A further decrease in concentration to 12.5 mmol/L results in a dramatic increase in drug retention. This effect could be due to a loss of buf fering capacity at such low phosphate concentration. Elution from the nonpolar column is dependent upon the ability of the buffer to stabilize the charge on these basic drugs. If this ability is lost, the nonpolar drugs remain on the hydrophobic column. A phosphate concentration of 25 mmol/L was selected for the assay protocol. Drug elution times were acceptable and stable from run to run. Phosphate precipitation should not be a problem at this concentration. Flowrate The mobile phase flowrate was varied from 0.5 to 2.0 ml /min in several steps to determine the affect on the elution of all the assay components. The drugs were chromatographed at the following flowrates: 2.0, 1.75, 1.5, 1.25, 1.0, 0.75, and 0.5 ml/min. 58 LU PHOSPHATE CONCENTRATION mmol/L concentration on retention. Fiq 19 Effect of phosphate buffer drug Components are: A, procainamide; B, NAPA; C, NPP; D, lidoc; E, NDAD; and J, propl. F, 4-OH propl; G, disop; H, quin; I, dihyq; 59 As theory predicts (18), an increase in the flowrate shortened the elution time (Figure 20). Elution order was maintained throughout the range of the flowrate tested. Drug resolution, however, as defined in Figure 21, decreased directly with flowrate. A comparison of pro cainamide with disopyramide showed the following: Flowrate (ml /min) Resolution 2.0 4.90 1.0 3.53 0.5 2.82 An improvement in resolution at higher flowrates is illustrated by NDAD and lidocaine. These drugs coelute at a flowrate of 0.5 ml /min, but they separate at 2.0 ml/min. Peak areas were inversely proportional to flowrate. In all cases, an increase in the flowrate by a factor of two, decreased the peak area by approximately one-half. Noninterfering, endogenous peaks from serum components followed the same pattern. This behavior was thought to be related to the residence time of the absorbing material in the detector flowcell. 1.0 ml/min was selected as the flowrate for the assay protocol. Run length was acceptable and the column head pressure of 3000 psi was within the tolerance of the column. Although drug resolution is better at 2.0 ml/min than at 1.0 ml/min, a 5400 psi column head pressure is generated at the higher flowrate. At a given flowrate, head pressure varies 15% depending on the condition of the filters in the flow system, the degree of deterioration of the pre-column, and the analytical col umn. At a flow of 2.0 ml/min, head pressure could exceed the maximum allowable system pressure of 6000 psi (9). This would result in system shutdown and possible column damage. Column Temperature 45 and 55 C to deter Drugs were chromatographed at ambient, 35 , , analysis. Flowrates of 1.0 and mine the affect of temperature on the determine if elution could 0.5 ml/min were used at each temperature to temperature with a low be improved by the use of a high column along mobile phase flowrate. At both flowrates, retention times were little affected by temper 35 on the of ature between ambient and C. (Ambient temperature day 22 23). Because room temperature fluctu assay was C) (Figures 22 and 20 20 30 between ates between and C, the variation in elution times 60 CM CO 00 CM CM o CM UJ OC co 0.5 1.0 1.5 2.0 FLOWRATE ml/min Fig. 20. Effect of mobile phase flowrate on drug elution. Components are: A, procainamide; B, NAPA; C, NPP; D, lidoc; E, NDAD; F, 4-OH propl; G, disop; H, quin; I, dihyq; and J, propl . 61 Rt2 ' Rtl R = wx + w2 Fig. 21. Equation for resolution of two components. R - resolution. (Rt? - R.,\ - difference in retention time between the two components, (retention time is measured at peak maximum). ((W, + Wp)/2) - average of peak widths measured at 10% above the baseline of each peak. 62 LU I LU OC COLUMN TEMP C temperature on elution. Mobile Fiq 22 Effect of column drug Components are: A, procainamide; phase flowrate 1.0 ml/min. 4-OH propl; G, disop; B, NAPA; C, NPP; D, lidoc; E, NDAD; F, H, quin: I, dihyq; and J, propl. 63 COLUMN TEMP UC Fig. 23. Effect of column temperature on drug elution. Mobile phase flowrate 0.5 ml/min. Components are: A, procainamide; B, NAPA; C, NPP;-D, lidoc; E, NDAD; F, 4-OH propl; G, disop; H, quin; I, dihyq; and J, propl. 64 and 35 C was considered especially important. The instrument is not capable of oven cooling, and when it is operated at ambient temperature the column oven is usually three degrees higher, due to heat generated by the instrument. However, the data indicate that changes in ambient temperature should not cause elution problems. 35 At temperatures over C, drug retention time decreases and resol 55 ution worsens. At C disopyramide and quinidine converge, as do NDAD, 35 lidocaine, and 4-OH propranolol. Increasing the temperature beyond C offers no advantage because of poor resolution. Ambient temperature was chosen for use in the assay. Isocratic Elution Isocratic elutions are generally reproducible and more readily useful for routine analysis. Therefore, all drugs were chromatographed in mobile phases of fixed composition to determine elution characteris tics. All assay drugs were run at the following mobile phase ratios of organic: phosphate: 70:30, 60:40, 50:50, 40:60, and 30:70. The normal run time of 25 min was increased to 30 min to permit elution of as many drugs as possible from the column. Isocratic elution at 30 - 40% organic modifier results in extreme tailing of the later eluting drugs and in poor separation of the earlier eluting components. Some drugs do not elute at all. Resolution of N- propionyl procainamide and propranolol, as determined by using the equa tion in Figure 21, is 1.59 at 40% organic. By comparison, the gradient elution program chosen for the assay protocol yields a R of 3.09 for the same two drugs. The retention times of these two drugs are approx imately the same in both run. This illustrates the poor resolution and efficiency of the 40% isocratic run. At 60 - 70% organic phase the peaks converge and have poor resolution (Figure 24). Interferences may coelute with the assay drugs, all of which elute within the first seven minutes. An isocratic run with 50% organic modifier would be effective for the analysis of the antiarrhythmic drugs other than procainamide and NAPA. The remaining drugs separate well and have good peak symmetry. Isocratic analysis is, however, not appropriate for the simultaneous study. A gradient system was assay of all antiarrhythmics in this therefore investigated. 65 oc 30 40 50 PERCENT ORGANIC IN MOBILE PHASE Elution of components Fig 24 Drug elution using isocratic analysis. assay organic/phosphate composition throughout the run. using mobile phase of fixed elute from the chromatograph A dashed line indicates that the component did not condition. Components are: at the indicated isocratic A, procainamide; B, NAPA; C, NPP; D, NDAD; E, 4-OH propl; F, disop; G, quin; H, dihyq; I, propl. 66 Gradient Development Several gradient programs were investigated to determine the best conditions for separation of all components in the assay. This was done adjustments in the by making length of time during which the mobile phase composition "slope" would change (i.e. the of the gradient), and by the changing organic/phosphate ratio at the beginning and end of the run. In all runs, the mobile phase composition is constant for the initial 2 min. At 2 min the linear gradient, described below, begins. The gradient reaches a final mobile phase composition at the times shown and this composition is maintained until 25.5 min. The mobile phase then returns to its initial proportion to equilibrate the column for 5 min before the next run is initiated. The slope of the gradient is defined as the change in percent organic divided by the time, in minutes, during which the change takes place. Experimental Linear Gradients Gradient Initial % Final % Grad ient Termination Slope Organic Organic % org/mi A 20 70 20 min 2.78 B 20 70 15 min 3.85 C 20 80 20 min 3.33 D 20 70 23 min 2.38 E 30 70 20 min 2.22 F 30 70 23 min 1.90 The slope of the gradient and initial percent organic are cri tical for adequate separation of all drugs. Steep slopes, such as 3.85% org/mi n in gradient B, do not resolve disopyramide and quinidine. Gradients using a higher initial percent organic, 30% in gradient E and gradient F, fail to adequately separate procainamide and NAPA. Gradients with intermediate slopes, using a 20% organic such as 2.78% org/min (gradient A), 3.33% org/min (gradient C), and 2.83% org/min (gradient D) provide the best resolution. Except for lidocaine and NDAD, all drugs are separated by at least 1.0 min. Gradient A resolved lidocaine and NDAD better than either gradients C or D'and was therefore adopted as the gradient of choice. Gradients are presented in Figures 25-30. METHOD OPTIMIZATION After the mobile phase conditions had been decided upon, refinements 67 '--' ii.il- : -f-- Hi.il ii i hi- iii !Hi! i.Hi . . . li H Xiii'r-ZZ \zzz ..--.. :--:-.i_i::: - X . - XE- t ii-E-- i.HHi " x~- !.:::.. -- ZZZZZ " """ fXpX ' - . "'ip.X . . X1XE . 1 X .._,.. XE ... . - ..". i r - - -.E-X- i .!![!-.! ..;-. X iiii 'iH -E- XE aaL -E. .;..-"-. ZAZ T , \ ;xfji" iiii!--" EiiE ii!! - . :xffxf 4. EfEfTXf ii!+- -- f.Xi--_ XEii-T i TTXEX o . . . zzzzzz: .__.J " - r co "1 j- . r --- "Z\" "7' " ~~ ii i hX .__ - --, - - - - T ii - X : . 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'.---_ z "iii- X.i- _._d ! 1 _ "I -H j i o * * 1 - XE 1 '.EXjEEX E - -. _ _.__ f^E \ iiHiJ iiii r .p. -^ ' 7-i ...ii:.. iiH " t . nil 1 ,- 1- - " * -j "-E . iH -xx-tIEi-i --"XT=JxX:X{EX yA \XiHiXX.*TTx} 1 EJ 12 16 20 24 TIME MIN. Fig. 25. Gradient elution. Change in percent organic of the mobile phase with time, during the run. Gradient A: initial % organic 20., final % organic 70, gradient termination 20 min, slope 2.78 % organic/min. Components are: A, procainamide; B, NAPA; C, NPP; D, lidoc; E, NDAD; F, 4-OH propl; G, disop; H, quin; I, dihyq; and J, propl. 68 00 Zd CO O L_ O CD OC o 12 16 20 24 TIME MIN. percent organic of the mobile phase Fig. 26. Gradient elution. Change in Gradient B: Initial % organic final % with time, during the run. 20, slope 3.85 % org/min. Components organic 70, gradient termination 15 min, lidoc and NDAD are: A, procainamide; B, NAPA; C, NPP; D, E, (coeluting) ; F, 4-0H propl; G, disop; H, quin; I, dihyq; and J, propl. 69 ~ Z"" " - - - - - ,. .. 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Components organic 80, gradient termination 20 min, lidoc and NDAD are: A, procainamide; B, NAPA; C, NPP; D, E, (coeluting); F, 4-OH propl; G, disop; H, quin; I, dihyq; and J, propl. 70 ..j 1 ' '( . - - ( I..: ": zzz - 'ZZ- 1 :- flXtHX ::fE-:.i -n.ir... .iiii in: ' .4 - - _..__ ii . .. - -zzz. _ ':-:-'- lid. -- ,-- - - -t- ^ - 'A - xtXT - .".; i r hh.1i n 1 - ' -- 7IEE T: XEf - ' ' id- "- Z- Z-.'Z -'.in AAA--. d 1 . . .4- - .- Hi' ' -"- - i-jn: -.- - - .! ... : 1 Ijll - - " x- i .-J nn. r: 'r o zA z d CO : " - zz. '' -t "1 - 7 ". ZZZZzA i . r '- i- r XffXE . r Z ip x p 1 " i --1- - 1 ; J 11 . HT . 7. d . : . HlXpi. J- hh:.*-- TEEnE Hi.r :_ eEE: - - -i in: -ii 1 zzzzz:.. . \ 'zzz . i | : : -;-tEl i - - : Hi -iiii! illjiE XT7 , EEETE . . p_nn - ;- |i ZZAZz TEpii TiXEX Zz E ffE , E--TX-- niiEi ... .iiji-1 -- EXfii A XXffiif-f EEf E TEx;f~TEEEX o H fEiXE 1 J i 1 1 1--. ' y">* 1 i i i : ; 1 EE n4.n - E| HlXiH 1.J-H- -Ei TEEf-.E ZZAzZZ. .zzHii i..n il- -Hii- ! 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Change in percent organic of the mobile phase with time, during the run. Gradient E: Initial % organic 30, final % organic 70, gradient termination 20 min, slope 2.22 % org/min. Components are: A, procainamide; B, NAPA; C, NPP; D, lidoc and E, NDAD (coeluting); F, 4-OH propl; G, disop; H, quin; I, dihyq; and J, propl. 72 ' " . - --.- f Z "\ i: i -. XJ I -- 1 - E :-- zzzz . XEA. ; f 1 1 1 1 ..\ ! - :.'- -j - iiiTii: l-H " TXXE-f EE T-H x"fx 1- '4 | ! i 1 ' . 1- r - T ..., 1 ' " TEE." ffEfE - - ' -- X~ '')-.- i- ---1 - f --ElEE ZZZZ.Z EX . !.T. 1 1 n Ei - x j zzzz :.- " i i- I"- ' 1" n-Htii X -X xL"7- i H 1 o - I - - 1 TX| XE CO . ._u . X.X'Efi ZZZZZZ zzz~ X JEfX i p EX TE. T _ - : 1 1-'. e :iX-fEEHt_ "i :E4fi]EEfi 1 in.j fi+Eff EiXfX: ZZZZZZZ ~zz i: i. n : 11 zzzizzzl zzzzz kj-x -r4~E "-TE E. T iff f ; zz'zLza ' . - zz. : - ' - | EEiffE AZ T- zzz: ir-fn.-i "-E- ii. : 4_X . -i.|.i .7 TXEX 4 Z Z .! 7 X zzz ' -__ T"-p!iE . 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E X ZC-ZZ "EEEf4.'.- E - id 1 i zzzzzzz. i -- , . .._! iXpx ..ZZ, . - - ..J . __ _ ,_j._ 1 ZAZZ. 1 1 1 "ZZZZZZZZZZ _zz. zzz: - -ZS - " E~" --. . .-. -. _ zzA-.zzz zzzz ; ._ . Z-Z-.2-.-ZZZ Z.ZZ-ZZ: IXE _ f.fxx.x.-11:1 !__. i ---.zizzz.- i 7Z7----- 1 1-1- - ---;-"ZZZZ.Z i zzz* _. . zz-zzz E "! f XEpT^.E - i-e_ -; ' ZZZZZZZ. ZZ]ZZZ 1 r: CM 1 ZZLZZ - i7EE 1 1- ZAzZ . zz AzLzz-z fpibfrX ZZZZ HiiLzzzzL h-EE T ' --.----- Hi- f 1 E _E E Z.Z. - : ! 1 : : 1 X EX 1:: X \ z E--X--i ffiT A 11 .1 A^zL ; 1 - ---' - ' ' -1 _4_hi-.ii --. __4 zz !..- 'Iii :_:.:. ii X_XJ."f "X - 1 : 1 ii in. 1 IH , J-fi . -: J . 1 \ 1 i--A i - ---.1 1 ! . 111111 _ X.TT ZZZZ- zZZZZZ. EXiErinf E <-> , [ 1 f- -e- ~ i._4 -. .. J-zzz - : 1 1- - zz:zz iiEi :Hi.iiH_TffffiEEE ____J - - - .. _L_. - - - !- . ! eeXe: - up- ZZZZZZZ-. E "-" f bxE:"-x:"" 1 ip___..pjEfilH "X - - l : i 4...-.J ^x .. _--. j ------i- -- -u- - 1 iH- z-ZZi 4 1 in ZZ H. ' -111.1- y__XJIX._E -id f_ L - . , .. . ..i. . . _ , , | ... ._i _i ' "* ' " -...Zp_XEJ."X-J:EE_EXiHiii .Hii "1 -f ii E fZ~ZZ.- ..TJ..~X."X. _.TJ-fXX , zzzzzzzz. i ZZ 1 0 8 12 16 20 24 TIME MIN. Fig. 30. Gradient elution. Change in percent organic of the mobile phase with time, during the run. Gradient F: Initial % organic 30, final % organic 70, gradient termination 23 min, slope 1.90 % org/min. Components are: A, procainamide; B, NAPA; C, NPP; D, NDAD; E, lidoc; F, 4-OH propl; G, disop; H. quin; I, dihyq; and J, propl. 73 were made in the instrument settings to select a detector wavelength where propranolols absorption would be maximum. This was necessary of because the drug's low - therapeutic serum level, (20 100 yg/ml ) . Ultraviolet absorption scans were performed. The necessary specimen and injection volumes to be used in the assay were also determined with the propranolol sensitivity requirements in mind. Wavelength For Analysis All drugs were prepared in mobile phase at a concentration of 5.0 mg/L. Each was then scanned in a Perkin-Elmer 559 UV/VIS spectropho tometer from 500 nm to 190 nm. The liquid chromatograph is also capable of scanning but the time required is considerably longer than with the Perkin-Elmer. A liquid chromatographic scan of propranolol (Figure 31) is included for illustrative purposes. The wavelength for analysis was chosen at propranolol's absorbance maximum of 212 nm. All other drugs have sufficient absorbance at this wavelength to allow accurate quantitation. A reference wavelength of 430 nm was selected because none of the drugs absorb at this wavelength. The data in Table 20 point out the advantage of a variable wavelength detector. Propranolol and lidocaine have over a 20 fold increase in absorptivity at 212 nm when compared to 254 nm. Seven of the other components have at least a two fold increase in absorptivity at the lower wavelength. Assay Volumes The assay volumes were selected to maximize the peak area of pro pranolol and thereby enhance its analytical sensitivity. The volume of mobile phase used for reconstitution after extraction was 150 yl . An injection volume of 75 yl was used. This volume allowed 50% of all extracted material to be injected into the chromatograph. This per centage is the maximum possible amount with the available vials and in jection system. The Clin-Elute CE-1003 column can handle a maximum of 3.0 ml of aqueous material without overloading the column matrix (19). By using 2.5 ml of serum and 200 yl of Na2C03 in the extraction procedure, 2.7 ml of aqueous material is placed onto the column. The volumes selected for use in the assay protocol maximize the amount of drug injected into the chromatograph. This should allow quantitation of 74 SCHN ULMftX 54B START SPL cr^ae_ 3BE - 4B0 45 -500 Fig. 31. HPLC ultraviolet absorbance scan of propranolol from 190 nm to 540 nm. Absorbance readings are taked every 2 nm. Wavelength (nm) is printed every 50 nm. 75 TABLE 20. ULTRAVIOLET ABSORBANCE SCANS AT SEVERAL WAVELENGTHS Absorbance Drugb 205 nm 210 nm 212 nm 220 nm 254 nm Procainamide .55 .46 .45 .27 .23 NAPA .61 .47 .40 .25 .35 NPP .54 .44 .37 .26 .30 NDAD .43 .32 .28 .20 .09 .16 .07 Disopyramide .45 .32 .28 .50 .35 Quinidine .74 .68 .61 .41 .23 Dihydroquinidine .61 .56 .52 .88 .75 .04 Propranolol .75 .88 Lidocaine >1\0 >1.0 >1.0 .60 .05 a Instrument sensitivity 1.0 AUFS. b Concentration (5 mg/L) in mobile phase. 76 propranolol in - the therapeutic range of 20 100 yg/L. Internal Standard Selection Procaine, P-chlorodisopyramide, and N-propionyl procainamide were evaluated as suitable internal standards in the assay. Procaine was rejected as an "tail" internal standard because of its tendency to under the chromatographic conditions of the assay. P-chlorodiso pyramide was also rejected. It eluted very late into the run (20 min) and had a only 74% extraction efficiency. It was also found to coelute with carbamazepine and potentially interfere with accurate quantification of propranolol (see Interference Study Table 9). N-propionylprocainamide proved to be a suitable internal standard. It elutes about midway into the run and has an 84% extraction efficiency. None of the forty- two drugs tested in the interference study elute within 2 min of NPP. NPP was adopted as the internal standard for the assay protocol . The amount of N-propionylprocainamide was selected in order to produce a peak area ratio of drug (or metabol ite)/internal standard as near to unity as possible when the drug is in the therapeutic range. The variation of absorptivities at 212 nm, and the expected amount of material extracted from serum for each analyte, produced a large range in the ratios. The area ratio at the mean concentration of the run- to-run precision study is shown in Table 6. Even at the small peak area ratio for propranolol, the microprocessor is able to compute the area precisely enough to achieve a relative standard deviation of 5.1%. DRUG EXTRACTION Several organic solvents were tested for their ability to extract the drugs of interest from serum. Once extracted, the chromatographic conditions developed previously were used to quantify the drugs. In the first experiment, drugs were isolated from serum by manual liquid- liquid extraction. In the second part of the study, the drugs were extracted with the use of the Clin-Elute CE-1003 columns. Manual Extraction A control of all antiarrhythmic drugs, at their therapeutic con centrations was prepared by spiking pooled serum that was known to be drug free. The control was added to test tubes, followed by 1.0 mol/L Na2C03 and the appropriate organic solvent. The Na2C03 is necessary 77 to adjust the serum to a high pH (approximately 10) where the basic drugs, existing in an unionized hydrophobic form, can be easily extracted into the nonpolar solvent. The tubes were vortexed and centrifuged. The organic layer was then transferred to a new test tube to evaporate the organic solvent. Each residue was reconstituted with 150 yl of mobile phase, vortexed, and removed to a microvial for chromatographic analysis. Solvents of various polarities were selected to test the drugs extractions (acetonitrile, chloroform, methylene chloride, hexane, heptane, N-butyl chloride, ethyl acetate, isopropanol, and toluene). of Each these were also tested with the addition of 2% isoamyl alcohol. Manual extraction proved to be a poor technique for the isolation of the drugs from serum. Some extraction tubes formed emulsions. Others produced a white turbid solution upon addition of the mobile phase. Long periods of time (30 min) were also required for eva poration of the extraction solvents. No useful information was gained from the manual extraction experiment. Clin-Elute Extraction Two types of Clin-Elute extractions were performed. The first employed only a single extraction step. The second utilized an acid back-extraction to transfer the basic drugs from the organic phase into a microvolume of aqueous HC1 . Single Extraction Clin-Elute CE-1003 columns were prepared by adding 200 yl of 1.0 mol/L Na2C03 and 50 yl of 0.2 mg/L N-propionylprocainamide to each column. Frozen serum controls of the antiarrhythmic drugs were thawed and 2.5 ml were then applied to appropriately labeled columns placed over 16x150 mm test tubes. Three additions of 4 ml of organic solvent were added to the columns. Each eluted solvent was evaporated to dryness at 50C. The residues were redissolved in 150 yl of mobile phase and 75 yl of this solution was to be injected into the chromatograph. The sol vents tested were acetonitrile, chloloform, methylene chloride, hex and toluene. ane, heptane, N-butylchloride, ethyl acetate, isopropanol, Each of these solvents was also tested with the addition of 2% isoamyl alcohol v/v to reduce nonspecific adsorption to glass (20). Isopropanol and acetonitrile (with of without isoamyl alcohol) would not pass through the column. The mobile phase reconstitution of the residues from the other solvent extractions produced milky suspensions. 78 To prevent possible damage to the system, the suspensions were not injected. Neutral extracting serum components may have been responsible for the suspensions. No useful extraction information was gained in this experiment. Dual Extraction Clin-Elute CE-1003 columns were prepared for extraction of basic drugs by the addition of 200 yl of 1.0 mol/L Na2C03 and 50 yl of 0.2 mg/L N-propionylprocainamide to each column. The eluent tube placed under each column had 200 yl of 0.1 N HC1 added to it. Frozen controls of antiarrhythmic drug in serum were thawed and 2.5 ml were applied to appropriately labeled Clin-Elute columns. Three addtions of 4 ml of organic solvent were then added to each column. The eluent tubes were each vortexed for 45 s to back-extract the basic drugs into the HC1 . Each tube was centrifuged for 10 min, the organic layer was aspirated and discarded, and the aqueous HC1 phase was dried under N?, after 300 yl of methanol had been added. The residue in each tube was then re constituted with 150 yl of mobile phase and 75 yl of this solution was injected into the chromatograph. The solvents tested were: meth ylene chloride, chloroform, ethyl acetate, toluene, hexane, heptane, N-butylchloride, methylene chloride:isopropanol (90:10), and methylene chloride: hexane (50:50). All of the above solvents and mixtures were also tested with the addition of 2% isoamyl alcohol. Residues from methylene chloride:isopropanol extractions formed slightly cloudy suspensions upon addition of mobile phase. All other extractions were clear and colorless. Solvents and mixtures which had densities less than the 0.1 N HC1 were easy to work with because the HC1 would remain as an easily dis cernible phase at the bottom of the test tube thus making aspiration of the organic a simple procedure. Solvents that were more dense than 0.1 N HC1 proved to be undesirable because the HC1 remained on top of the organic phase which made aspiration difficult. It was not possible to aspirate the organic phase from extractions that had solvent densi ties near 1.00 gm/cc because no separation of phases occurred after centrifugation (e.g. methylene chloride:hexane). Solvents with den sities of less than 0.99 gm/cc should be used to avoid these aspiration problems. As shown in Table 21, the more polar drugs, such as procainamide 79 TABLE 21. CLIN-ELUTE EXTRACTION EFFICIENCIES. PERCENT RECOVERY OF ASSAY DRUGS USING THE ESTABLISHED DXTRACTION PROTOCOL WITH VARIOUS ORGANIC SOLVENTS. Solvent PA NAPA NPP NDAD DISOP QUIN DIHYQ PROPL Ethyl acetate 112 117 98 109 105 102 114 26 IAAa Ethyl acetate: 113 119 97 105 107 104 114 16 Toluene 8 <1 2 1 66 90 74 115 Toluene; IAA 53 18 50 84 106 106 112 100 Hexane <1 <1 <1 <1 <1 <1 <1 62 Hexane: IAA <1 <1 <1 <1 88 104 109 108 Heptane <1 <1 <1 <1 <1 <1 <1 <1 Heptane: IAA <1 <1 <1 <1 86 102 106 107 Butyl chloride 14 <1 5 <1 74 78 67 101 76 Butyl chlorides IAA 39 1 49 49 90 86 85 37 <1 Methylene chloride: 114 112 75 84 18 49 c 27 <1 Methylene chloride: IPA. IAA 112 104 66 78 16 38 56 Methylene chloride 107 101 92 70 66 91 89 94 78 22 Methylene chloride.iIAA 114 113 91 103 56 67 Chloroform 112 110 94 97 60 90 80 52 19 Chloroforms IAA 105 107 85 93 44 66 Methylene chloride;hexane 73 85 102 103 102 105 70 Assay protocol solvent 96 solvent: IAA is 98:2). a IAA-isoamyl alcohol (ratio of chloride:IPA is 90:10) b IPA-isopropyl alcohol (ratio of methylene c chloride:IPA:IAA is 88:10:2) Ratio of methylene d separation did not occur. Not assayed because phase e 49:49:2. Methylene chloride:hexane:IAA 80 and NAPA, extract well into the slightly nonpolar solvents, such as chloroform. Nonpolar drugs, like propranolol, extract best into very nonpolar solvents, such as toluene or hexane. The solvent system adop ted, methylene chloride:hexane:isoamyl alcohol (49:49:2), takes advan tage of the good recovery of propranolol by hexane- isoamyl alcohol and the good recovery of the other drugs by methylene chloride. In addition, the mixture has a density of 0.989 gm/cc, which ensures that the HC1 will remain on the bottom of the tube after centrifugation. 81 CONCLUSION We have developed a high performance liquid chromatographic assay that is of adequate sensitivity and precision for clinical use. The "family" extraction and resolution of both drugs and metabolites of a of pharmacological agents (cardioactive drugs) from spiked sera, allows the laboratory to use a standard procedure regardless of which drug or drugs are requested for analysis. The 30 min chromatographic run is not a major drawback because of the autosampling feature of the chrom atograph that allows the assay to be performed (after extraction) with out operator intervention. The assay has only a limited number of interferences because most acidic and neutral drugs are removed by the extraction procedure. Al though carbamazepine and glutethimide may interfere with the quantifi cation of propranolol, they can be removed by additional organic ex tractions if the laboratory is made aware of their presence. Quinine and lidocaine cannot be eliminated as interfering agents by the same technique due to their basic character. Because it is impossible to analytically test all drugs and metabolites for interference, we are closely scrutinizing all patient chromatograms for unidentified peaks. This specific and accurate liquid chromatographic assay does not suffer from the many interferences found in manual fluorometric and colorimetric methods. HPLC compares favorably with enzumeimmunoassay of It in its ability to accurately quantitate the drugs interest. also provides a means of monitoring some major metabolites which, with increased knowledge of drug action, may be clinically useful. The extensive work performed on the development of the chromato of graphic conditions will be valuable in the implementation future tested as inter assays on this instrument. Several drugs which were the established ferences are being considered for assay, utilizing chromatographic conditions. 82 REFERENCE LIST 1. Bigger, Jr., J.T., and Hoffman, B.F., Antiarrhythmic drugs, in "The Pharmacological Basis of Therapeutics" A.G. Gilman, L.S. Goodman and A. Gilman Eds. MacMillan Publishing, New York, (1980) p 761-792. 2. jjalle, and T., Gaffney, T., Propranolol Metabolism in Man and Dog. Mass Spectrometry Identification of Six New Metabolites. J. Pharma col Exp. Ther. 182, 83-92 (1972). 3. and Cramer, G., Isaksson, B., Quantitative Determination of Quinidine in plasma. B. Scand. J. Clin Lab. Invest. 15, 553-556 (1963). 4. Shand, D.G., Nuckolls, E.M., and Oates, J. A., Plasma Propranolol levels in adults with Observations in Four Children. Clin. Pharma col. Ther. U, 112-120 (1970). 5. 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