515
Clinica Chimica Acta, 69 (1976) 515-524 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CGA 7864
THE SIMULTANEOUS DETERMINATION OF LIDOCAINE AND PROCAINAMIDE IN SERUM BY USE OF HIGH PRESSURE LIQUID CHROMATOGRAPHY
REGINALD
F. ADAMS,
The Perkin-Elmer
(Received
January
FRANK
Corporation,
L. VANDEMARK
Main Avenue,
and G. SCHMIDT
Norwalk,
Conn.
06856
(U.S.A.)
20, 1976)
Summary This report describes an accurate, sensitive rapid procedure for the determination of lidocaine and procainamide at therapeutic concentrations in serum. The drugs and an added internal standard (procaine) are extracted from serum using charcoal adsorption. The analysis is carried out by high pressure liquid chromatography on a reverse-phase column using buffered aqueous acetonitrile as the mobile phase. Chromatography is complete in 10 min. C.V. values of 10% and 6% for concentrations of 1 mg/l and 20 mg/l, respectively, are attainable routinely. Total analysis time is 15 min.
Introduction Procainamide and lidocaine are important drugs for the treatment of cardiac arrhythmias. Plasma concentrations of the drugs relate more accurately than dosage to the clinical response of subjects requiring this type of therapy [ 1,2]. For the determination of procainamide and lidocaine, gas chromatography is often used [3,4] generally requiring different operating parameters for each drug. Fluorimetric [ 21 and spectrophotometric [ 51 procedures are additionally available for the determination of procainamide. Whatever method is used, it is essential that it possess good precision and accuracy especially as the therapeutic ranges for the drugs are narrow (procainamide, 4-8 mg/l [l] ; lidocaine, l6 mg/l L61). High-pressure liquid chromatography offers the advantages of a high resolution separation technique and a non-destructive detection system. In the present paper, a rapid extraction of the drugs by charcoal adsorption [8] from the sample matrix is followed by isocratic elution from a reverse-phase column. The mobile phase is buffered, aqueous acetonitrile. Compounds eluted from
516
the column are detected by their ultra-violet absorption at 205 nm. Parameters used permit separation of lidocaine, procainamide and an internal standard (procaine) simultaneously. The procedures are accurate, rapid and suitable for the routine determination of these drugs. Materials and Methods Reagents Lidocaine. As the hydrochloride, Sterling-Winthrop Research Institute, Rensselaer, N.Y. 12144. Proc~n~ide. As the hydrochloride, Squibb and Sons, E.R., Princeton, N.J. 08540. Procaine. As the hydrochloride, Sterling-Winthrop Research Institute, Rensselaer, N.Y. 12144. Acetonitrile. Ultraviolet grade, distilled in glass, Burdick and Jackson Laboratories, Inc., Muskegon, Mich. 49442. Methanol. Spectroquality grade. Dichloromethane. Spectroquality grade. Potassium phosphate dibasic. Analytical reagent grade. Potassium phosphate monobasic. Analytical reagent grade. Ammonium hydroxide. 28%, analytical reagent grade. Charcoal. Norit A, Neutral, Amend Drug and Chemical Co., Irvington, N.J. 07111. Standard drug solutions Lidocaine. 25 mg/50 ml. Dissolve 25 mg lidocaine hydrochloride in methanol and dilute to 50 ml. Procainamide. 25 mg/50 ml. Dissolve 25 mg procainamide hydrochloride in methanol and dilute to 50 ml. Procaine (internal standard). 25 mgf50 ml. Dissolve 25 mg procaine hydrochloride in methanol and dilute to 50 ml. Phosphate buffer. 0.2 M, pH 6.0. 61.5 ml, 0.2 M dipotassium hydrogen phosphate mixed with 438.5 ml 0.2 M potassium dihydrogen phosphate and the pH adjusted to 6.0. 2% Ammonium hydroxide. The stock reagent is diluted with deionized water. Acetonitrile/phosphate mobile phase. 10 volumes acetonitrile in 90 volumes 0.2 M phosphate buffer.
Culture tubes. 16 mm X 75 mm, screw cap, polytetrafluoroethylene lined, Kimble No. 45066-A. Culture tubes. 10 mm X 75 mm, disposable Kimble No. 73000. Bench centrifuge. Heating block set to 60” C. Vortex-type mixer. Evaporation manifold using dry air or nitrogen. Liquid chromatograph Perkin-Elmer Model 601 equipped with Model LC 55
517
detector, a 10 mV recorder, and a reverse-phase column, Sil-X-I. Data reduction. PEP-II Processor. Operating conditions for the chromatograph were: Mobile phase flow rate (ml/min, at 800 psi) 2 Colum oven temperature (“C) 40 Detector (nm, 0.2 aufs.) 205
0.5 m X 2.6 mm ODS-
Method 1. Transfer
to a 16 mm X 75 mm culture tube 1 ml serum, 10 ~1 internal standard (= 5 pg), 2 ml 2% ammonium hydroxide and approximately 8 mg charcoal. 2. Cap the tube and mix the contents vigorously for 15 seconds with a vortex-type mixer. Centrifuge 1 min at 2500 rpm. 3. Aspirate off the aqueous phase. 4. Add 1 ml dichloromethane to the tube. Repeat step 2. 5. Decant solvent into a 10 mm X 75 mm tube. 6. Evaporate the solvent at 60°C using a gentle air or nitrogen flow (rate not greater than 50 ml/min). The residue is dissolved in 20 ~1 methanol and 4 1-11 are injected into the liquid chromatograph. Experimental resdts A mixture of the pure drugs consisting of 1 pg each lidocaine, procainamide and procaine was applied to the column to determine the efficiency of the chromatography system. The chromatogram obtained is given in Fig. 1. The elution order of the compounds was procainamide, procaine and lidocaine and the overall analysis time was 10 min. Drug-free sera with added drugs were taken through the complete procedure. ‘A chromatogram of a serum extract is shown in Fig. 2. The drugs were added to the serum at a concentration of 1 mg/l. Identification of each compound was by correspondence of retention time with that of a pure standard. Sera from subjects on lidocaine or procainamide therapy were analyzed by the procedures. Resulting peaks were identified by retention time. Quantitation was by computer using peak areas of the drugs compared with peak areas obtained from extracted sera with known concentrations of lidocaine and procainamide. The internal standard peak area was used as a correction for extraction and injection variations. Peak area measurements were assessed for linearity by extracting from sera known concentrations of the compounds. Four concentrations of each drug were used: 0.1,1.0,10 and 20 mg/l. Four aliquots of each concentration were extracted and peak areas plotted against concentration. Results are given in Fig. 3. A linear relationship was obtained for the range O.l20 mg/l. Fig. 4 illustrates three chromatograms. The first (on the left) is that of a known drug-free serum taken through the procedures without added internal standard and shows the effectiveness of the extraction procedure in providing a background relatively free from unwanted peaks. The remaining two chromatograms are from a serum of a subject on procainamide therapy, and from a
Fig. 1. Standard
chromatogram.
Fig. 2. Chromatogram each drug.
of serum
1 pg each compound with added
injected
drugs taken
onto the column.
through
the procedure.
601
LINEARITY EXTRACTED
50 -
SAMPLES
1 mg/l
1 d
PLOT (l.Oml
Concentrations:
ALIQUOTS)
cn 40-
Y a
20-
I 0
20
10
1
mg /LITER Fig. 3. Linearity caine.
plot.
Peak areas plotted
against serum concentration
of lidocaine.
procainamide
and pro-
519
J
Fig. 4. Chromatograms of three serum extracts. The first chromatogram (on left) is of a known drug-free serum and illustrates a typical serum background. The second chromatogram was from serum containing procainamide (2.5 me/l); the third ehromatogram was obtained from serum where lidocaine was calculated to be 2.6 mg/l.
TABLE
I
RECOVERIES Calculated centration
(%)
from serum-based were analyzed.
samples
taken
through
Concentration
1 mgll
20 mg/l
65 i 3 64 f 3 72 f 4
68 f 3 64 * 3 12 + 2
-__
Lidocaine Procaine F’rocainamide
* N = 4 samples each concentration.
the complete
procedure.
Four
aliquots
of each con-
520
Fig. 5. Two chromatograms from sera of subjects on procainamide therapy. Concentrations were calculated to be 0.8 ancl 3.1 mg/l, respectively. The first chromatogram (on left) contained a peak eluting immediately before procainamide with a retention time equivalent to N-acetyl-procainamide.
serum of a subject on lidocaine therapy. The procainamide concentration was calculated to be 2.5 mg/l; that of lidocaine was calculated to be 2.6 mg/l. Fig. 5 shows the chromatograms obtained from two serum extracts. Procainamide was calculated to be 0.8 mg/l and 3.1 mg/l, respectively. In the first chromatogram, the peak eluting immediately before procainamide was tenta-
521
tively characterized as the N-acetylated metabolite of procainamide spondence of retention time with the synthesized metabolite.
by corre-
Recoveries Recovery data were collected by adding procainamide and lidocaine to known drug-free sera, taking aliquots of the sera through the complete procedures and comparing peak areas of extracted drugs with peak areas of pure standards. The results for 4 aliquots of each concentration of the drugs at 1 mg/l and 20 mg/l concentration are summarized in Table I. Recoveries at a concentration of 1 mg/l were 65 ?r 3%, 64 _+3%, 72 f 4% for lidocaine, procaine and procainamide, respectively. At 20 mg/l, recoveries were 68 + 3%, 64 i: 3%, 72 f 2%, respectively, for lidocaine, procaine and procainamide. Precision data were obtained by taking aliquots of two serum pools containing added drugs to give concentrations of 1 mg/l and 20 mg/l, respectively. Single aliquots of each pool were processed daily after addition of the internal standard for twenty working days. The reproducibility was acceptable: 1 mg/l: lidocaine, C.V.% 10.6; procaine, C.V.% 9.8; procainamide, C.V.% 8.2. At 20 mg/l, the results were: lidocaine, C.V.% 5.6; procainamide, C.V.% 5.6. These results are summarized in Table II. Sensitivity The sensitivity of the method is limited largely by sample size, extraction efficiency and background. With the recommended 1 ml sample size and the use of the special grade solvents, the sensitivity is adequate to permit detection of the drugs in concentration as small as 0.1 mg/l. Accuracy To assess accuracy, the procedures were compared with other methods. Fifteen sera from subjects on lidocaine therapy were analyzed for lidocaine both by an accepted gas chromatographic method [ 41 and by the liquid chromatographic procedure. In general, the values showed a good correlation. The results are summarized in Table III. The coefficient of correlation was 0.989; slope, 0.983; Y-intercept, 0.05. TABLE
II
DAY-TO-DAY Calculated
PRECISION from
aliquots
DATA of
two
serum
pools
containing
Concentration
1 mg/1
20 mg/1
Lidocaine w S.D. C.V.
0.94 0.10 10.6
21.4 1.2 5.6
Procainamide x
0.98
S.D.
0.08
1.1
C.V.
8.2
5.6
19.6
1 mg/l
and
20 mg/l
drug
concentrations.
N = 20.
522
TABLE
III
LIDOCAINE
COMPARISON
Results
obtained
by
relation
= 0.989,
slope
comparing
Sample
No.
duplicate
= 0.983,
Lidocaine
_
_~__..
samples
y-intercept
cont.
analyzed
by parallel
method.
N = 15.
Coefficient
of cor-
= 0.05.
(mg/l)
GC
LC 15.8
1
16.7
2
15.2
15.9
3
13.8
14.1
4
12.1
10.4
5
10.8
11.6
6
9.2
a.7
7
7.9
8.3
8
6.8
6.9
9
6.2
6.9
10
5.9
5.4
11
5.8
5.0
12
5.7
5.9
13
4.2
3.7
14
2.4
2.5
15 ___~~_
1.8
2.0
A spectrophotometric procedure, based on the absorption difference at 272 nm between acid and alkali solutions of procainamide [ 5 J was used as a comparison method. Twelfve clinical sera were analyzed by the two methods. Results are summarized in Table IV. The coefficient of correlation was calculated to be 0.983; slope, 0.643, Y-intercept, 1.44. Interferences
Other
TABLE
drugs were evaluated
Results
Sample
with the analysis of
obtained slope No.
COMPARISON by
= 0.643,
comparing y-intercept
duplicate
samples
= 1.44.
Procainamide
concn.
uv
LC
1
16.2
13.1
2
10.8
a.9
3
9.0
9.7
4
6.4
6.8
5
5.0
4.6
6
3.2
2.8
7
2.9
3.4
8
2.4
2.5
9
1.7
2.1
10
1.7
1.5
11
0.5
0.3
12
0.4
0.3
__..
interferences
IV
PROCAINAMIDE
0.983,
as potential
~.~~
~_~
(mg/l)
by parallel
method.
N = 12.
Coefficient
of correlation
=
523
TABLEV INTERFERENCES Data on retention
times obtained Retention
Ethosuximide Procainamide Procaine Primidone Lidocaine Phenobarbital Amobarbital Secobarbital Pentobarbital Propoxyphene Glutethimide Methaqualone
by cbromatographing ~__
pure standard
solutions.
time @in)
3.4 4.0 5.8 6.4 7.8 9.0 >12.0 >12.0 >12.0 >12.0 >12.0 >12.0
lidocaine and procainamide. Pure drug solutions were chromatographed individually and retention times of each noted. Drugs tested were ethosuximide, primidone, phenobarbital, amobarbital, pentobarbital, secobarbital, propoxyphene, glutethimide and methaqualone. Ethosuximide was found to elute at 3.4 min, before procainamide (4 min). Primidone eluted at 6.4 min, after procaine (5.8 min) and before lidocaine (7.8 min). Phenobarbital eluted at 9 min, after lidoCaine (7.8 min). The other drugs tested eluted after 12 min. None of the tested compounds constituted an interference under the conditions of the analysis. These results are summarized in Table V. Discussion
The value of regular measurements of serum or plasma concentrations of drugs used for treatment of cardiac arrhythmias has been well established [ 71. The therapeutic concentrations of lidocaine and procainamide pose special problems to the analyst: special columns have been recommended for gas chromatographic procedures [ 91; relatively large sample sizes; different analytical procedures are generally recommended for the individual drugs. The data presented in this report indicates that high pressure liquid chromatography using one set of operating conditions is suitable for the determination of lidoCaine and procainamide in serum. The sensitivity, precision and accuracy are satisfactory. For those samples analyzed by parallel methods, the correlation was good. Detection by absorption at 205 nm gives adequate sensitivity but poor selectivity. This lack of selectivity is largely offset by the chromatographic separation. A useful feature of the detection system is that eluted analytes may be recovered for additional investigation. The analysis time for a single sample is about 15 min, including extraction and chromatography. The use of procaine as an internal standard has advantages because of its retention time and extraction characteristics. Some sera may contain significant amounts of procaine hydrolase. Therefore sera should be extracted promptly after addition of the internal standard. In conclusion, the high pressure liquid chromatographic procedure for the
524
routine determination to other procedures.
of lidocaine
and procainamide
offers a useful alternative
References 1 2 3 4
Vesell, ES. and Passananti, Koch-Messer, J. and Klein, Atkinson, A.J. (1972) Clin. Aggarwal, V. and Bath, R. Press, Cleveland, Ohio 5 Barnhardt, F.E. (1975) in land, Ohio
G.T. (1971) Clin. Chem. 17,851 S.W. (1971) Am. Med. Assoc. 215, 1454 Chem. 18.643 (1975) in Methodology for Analytical Toxicology Methodology
for Analytical
Toxicology
(Sunshine,
(Sunshine,
I., ed.), CRC
I. ed.). CRC Press, Cleve-
6 Strong, J.M. and Atkinson, A.J. (1972) Anal. Chem. 44.2287 7 Bayes, R.N., Scott, D.B., Jebson. P.J., Goodman. M.J. and Julian, D.G. (1971) Clin. Pharmacol. Ther. 12,105 8 Meola, J. and Vanko, M. (1974) Clin. Chem. 20, 184 9 Meola, J. (1975) in Methodology for Analytical Toxicology (Sunshine, I., ed.), CRC Press, Cleveland, Ohio
Clinica Chimica Acta, 69 (1976) 515-524 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CGA 7864
THE SIMULTANEOUS DETERMINATION OF LIDOCAINE AND PROCAINAMIDE IN SERUM BY USE OF HIGH PRESSURE LIQUID CHROMATOGRAPHY
REGINALD
F. ADAMS,
The Perkin-Elmer
(Received
January
FRANK
Corporation,
L. VANDEMARK
Main Avenue,
and G. SCHMIDT
Norwalk,
Conn.
06856
(U.S.A.)
20, 1976)
Summary This report describes an accurate, sensitive rapid procedure for the determination of lidocaine and procainamide at therapeutic concentrations in serum. The drugs and an added internal standard (procaine) are extracted from serum using charcoal adsorption. The analysis is carried out by high pressure liquid chromatography on a reverse-phase column using buffered aqueous acetonitrile as the mobile phase. Chromatography is complete in 10 min. C.V. values of 10% and 6% for concentrations of 1 mg/l and 20 mg/l, respectively, are attainable routinely. Total analysis time is 15 min.
Introduction Procainamide and lidocaine are important drugs for the treatment of cardiac arrhythmias. Plasma concentrations of the drugs relate more accurately than dosage to the clinical response of subjects requiring this type of therapy [ 1,2]. For the determination of procainamide and lidocaine, gas chromatography is often used [3,4] generally requiring different operating parameters for each drug. Fluorimetric [ 21 and spectrophotometric [ 51 procedures are additionally available for the determination of procainamide. Whatever method is used, it is essential that it possess good precision and accuracy especially as the therapeutic ranges for the drugs are narrow (procainamide, 4-8 mg/l [l] ; lidocaine, l6 mg/l L61). High-pressure liquid chromatography offers the advantages of a high resolution separation technique and a non-destructive detection system. In the present paper, a rapid extraction of the drugs by charcoal adsorption [8] from the sample matrix is followed by isocratic elution from a reverse-phase column. The mobile phase is buffered, aqueous acetonitrile. Compounds eluted from
516
the column are detected by their ultra-violet absorption at 205 nm. Parameters used permit separation of lidocaine, procainamide and an internal standard (procaine) simultaneously. The procedures are accurate, rapid and suitable for the routine determination of these drugs. Materials and Methods Reagents Lidocaine. As the hydrochloride, Sterling-Winthrop Research Institute, Rensselaer, N.Y. 12144. Proc~n~ide. As the hydrochloride, Squibb and Sons, E.R., Princeton, N.J. 08540. Procaine. As the hydrochloride, Sterling-Winthrop Research Institute, Rensselaer, N.Y. 12144. Acetonitrile. Ultraviolet grade, distilled in glass, Burdick and Jackson Laboratories, Inc., Muskegon, Mich. 49442. Methanol. Spectroquality grade. Dichloromethane. Spectroquality grade. Potassium phosphate dibasic. Analytical reagent grade. Potassium phosphate monobasic. Analytical reagent grade. Ammonium hydroxide. 28%, analytical reagent grade. Charcoal. Norit A, Neutral, Amend Drug and Chemical Co., Irvington, N.J. 07111. Standard drug solutions Lidocaine. 25 mg/50 ml. Dissolve 25 mg lidocaine hydrochloride in methanol and dilute to 50 ml. Procainamide. 25 mg/50 ml. Dissolve 25 mg procainamide hydrochloride in methanol and dilute to 50 ml. Procaine (internal standard). 25 mgf50 ml. Dissolve 25 mg procaine hydrochloride in methanol and dilute to 50 ml. Phosphate buffer. 0.2 M, pH 6.0. 61.5 ml, 0.2 M dipotassium hydrogen phosphate mixed with 438.5 ml 0.2 M potassium dihydrogen phosphate and the pH adjusted to 6.0. 2% Ammonium hydroxide. The stock reagent is diluted with deionized water. Acetonitrile/phosphate mobile phase. 10 volumes acetonitrile in 90 volumes 0.2 M phosphate buffer.
Culture tubes. 16 mm X 75 mm, screw cap, polytetrafluoroethylene lined, Kimble No. 45066-A. Culture tubes. 10 mm X 75 mm, disposable Kimble No. 73000. Bench centrifuge. Heating block set to 60” C. Vortex-type mixer. Evaporation manifold using dry air or nitrogen. Liquid chromatograph Perkin-Elmer Model 601 equipped with Model LC 55
517
detector, a 10 mV recorder, and a reverse-phase column, Sil-X-I. Data reduction. PEP-II Processor. Operating conditions for the chromatograph were: Mobile phase flow rate (ml/min, at 800 psi) 2 Colum oven temperature (“C) 40 Detector (nm, 0.2 aufs.) 205
0.5 m X 2.6 mm ODS-
Method 1. Transfer
to a 16 mm X 75 mm culture tube 1 ml serum, 10 ~1 internal standard (= 5 pg), 2 ml 2% ammonium hydroxide and approximately 8 mg charcoal. 2. Cap the tube and mix the contents vigorously for 15 seconds with a vortex-type mixer. Centrifuge 1 min at 2500 rpm. 3. Aspirate off the aqueous phase. 4. Add 1 ml dichloromethane to the tube. Repeat step 2. 5. Decant solvent into a 10 mm X 75 mm tube. 6. Evaporate the solvent at 60°C using a gentle air or nitrogen flow (rate not greater than 50 ml/min). The residue is dissolved in 20 ~1 methanol and 4 1-11 are injected into the liquid chromatograph. Experimental resdts A mixture of the pure drugs consisting of 1 pg each lidocaine, procainamide and procaine was applied to the column to determine the efficiency of the chromatography system. The chromatogram obtained is given in Fig. 1. The elution order of the compounds was procainamide, procaine and lidocaine and the overall analysis time was 10 min. Drug-free sera with added drugs were taken through the complete procedure. ‘A chromatogram of a serum extract is shown in Fig. 2. The drugs were added to the serum at a concentration of 1 mg/l. Identification of each compound was by correspondence of retention time with that of a pure standard. Sera from subjects on lidocaine or procainamide therapy were analyzed by the procedures. Resulting peaks were identified by retention time. Quantitation was by computer using peak areas of the drugs compared with peak areas obtained from extracted sera with known concentrations of lidocaine and procainamide. The internal standard peak area was used as a correction for extraction and injection variations. Peak area measurements were assessed for linearity by extracting from sera known concentrations of the compounds. Four concentrations of each drug were used: 0.1,1.0,10 and 20 mg/l. Four aliquots of each concentration were extracted and peak areas plotted against concentration. Results are given in Fig. 3. A linear relationship was obtained for the range O.l20 mg/l. Fig. 4 illustrates three chromatograms. The first (on the left) is that of a known drug-free serum taken through the procedures without added internal standard and shows the effectiveness of the extraction procedure in providing a background relatively free from unwanted peaks. The remaining two chromatograms are from a serum of a subject on procainamide therapy, and from a
Fig. 1. Standard
chromatogram.
Fig. 2. Chromatogram each drug.
of serum
1 pg each compound with added
injected
drugs taken
onto the column.
through
the procedure.
601
LINEARITY EXTRACTED
50 -
SAMPLES
1 mg/l
1 d
PLOT (l.Oml
Concentrations:
ALIQUOTS)
cn 40-
Y a
20-
I 0
20
10
1
mg /LITER Fig. 3. Linearity caine.
plot.
Peak areas plotted
against serum concentration
of lidocaine.
procainamide
and pro-
519
J
Fig. 4. Chromatograms of three serum extracts. The first chromatogram (on left) is of a known drug-free serum and illustrates a typical serum background. The second chromatogram was from serum containing procainamide (2.5 me/l); the third ehromatogram was obtained from serum where lidocaine was calculated to be 2.6 mg/l.
TABLE
I
RECOVERIES Calculated centration
(%)
from serum-based were analyzed.
samples
taken
through
Concentration
1 mgll
20 mg/l
65 i 3 64 f 3 72 f 4
68 f 3 64 * 3 12 + 2
-__
Lidocaine Procaine F’rocainamide
* N = 4 samples each concentration.
the complete
procedure.
Four
aliquots
of each con-
520
Fig. 5. Two chromatograms from sera of subjects on procainamide therapy. Concentrations were calculated to be 0.8 ancl 3.1 mg/l, respectively. The first chromatogram (on left) contained a peak eluting immediately before procainamide with a retention time equivalent to N-acetyl-procainamide.
serum of a subject on lidocaine therapy. The procainamide concentration was calculated to be 2.5 mg/l; that of lidocaine was calculated to be 2.6 mg/l. Fig. 5 shows the chromatograms obtained from two serum extracts. Procainamide was calculated to be 0.8 mg/l and 3.1 mg/l, respectively. In the first chromatogram, the peak eluting immediately before procainamide was tenta-
521
tively characterized as the N-acetylated metabolite of procainamide spondence of retention time with the synthesized metabolite.
by corre-
Recoveries Recovery data were collected by adding procainamide and lidocaine to known drug-free sera, taking aliquots of the sera through the complete procedures and comparing peak areas of extracted drugs with peak areas of pure standards. The results for 4 aliquots of each concentration of the drugs at 1 mg/l and 20 mg/l concentration are summarized in Table I. Recoveries at a concentration of 1 mg/l were 65 ?r 3%, 64 _+3%, 72 f 4% for lidocaine, procaine and procainamide, respectively. At 20 mg/l, recoveries were 68 + 3%, 64 i: 3%, 72 f 2%, respectively, for lidocaine, procaine and procainamide. Precision data were obtained by taking aliquots of two serum pools containing added drugs to give concentrations of 1 mg/l and 20 mg/l, respectively. Single aliquots of each pool were processed daily after addition of the internal standard for twenty working days. The reproducibility was acceptable: 1 mg/l: lidocaine, C.V.% 10.6; procaine, C.V.% 9.8; procainamide, C.V.% 8.2. At 20 mg/l, the results were: lidocaine, C.V.% 5.6; procainamide, C.V.% 5.6. These results are summarized in Table II. Sensitivity The sensitivity of the method is limited largely by sample size, extraction efficiency and background. With the recommended 1 ml sample size and the use of the special grade solvents, the sensitivity is adequate to permit detection of the drugs in concentration as small as 0.1 mg/l. Accuracy To assess accuracy, the procedures were compared with other methods. Fifteen sera from subjects on lidocaine therapy were analyzed for lidocaine both by an accepted gas chromatographic method [ 41 and by the liquid chromatographic procedure. In general, the values showed a good correlation. The results are summarized in Table III. The coefficient of correlation was 0.989; slope, 0.983; Y-intercept, 0.05. TABLE
II
DAY-TO-DAY Calculated
PRECISION from
aliquots
DATA of
two
serum
pools
containing
Concentration
1 mg/1
20 mg/1
Lidocaine w S.D. C.V.
0.94 0.10 10.6
21.4 1.2 5.6
Procainamide x
0.98
S.D.
0.08
1.1
C.V.
8.2
5.6
19.6
1 mg/l
and
20 mg/l
drug
concentrations.
N = 20.
522
TABLE
III
LIDOCAINE
COMPARISON
Results
obtained
by
relation
= 0.989,
slope
comparing
Sample
No.
duplicate
= 0.983,
Lidocaine
_
_~__..
samples
y-intercept
cont.
analyzed
by parallel
method.
N = 15.
Coefficient
of cor-
= 0.05.
(mg/l)
GC
LC 15.8
1
16.7
2
15.2
15.9
3
13.8
14.1
4
12.1
10.4
5
10.8
11.6
6
9.2
a.7
7
7.9
8.3
8
6.8
6.9
9
6.2
6.9
10
5.9
5.4
11
5.8
5.0
12
5.7
5.9
13
4.2
3.7
14
2.4
2.5
15 ___~~_
1.8
2.0
A spectrophotometric procedure, based on the absorption difference at 272 nm between acid and alkali solutions of procainamide [ 5 J was used as a comparison method. Twelfve clinical sera were analyzed by the two methods. Results are summarized in Table IV. The coefficient of correlation was calculated to be 0.983; slope, 0.643, Y-intercept, 1.44. Interferences
Other
TABLE
drugs were evaluated
Results
Sample
with the analysis of
obtained slope No.
COMPARISON by
= 0.643,
comparing y-intercept
duplicate
samples
= 1.44.
Procainamide
concn.
uv
LC
1
16.2
13.1
2
10.8
a.9
3
9.0
9.7
4
6.4
6.8
5
5.0
4.6
6
3.2
2.8
7
2.9
3.4
8
2.4
2.5
9
1.7
2.1
10
1.7
1.5
11
0.5
0.3
12
0.4
0.3
__..
interferences
IV
PROCAINAMIDE
0.983,
as potential
~.~~
~_~
(mg/l)
by parallel
method.
N = 12.
Coefficient
of correlation
=
523
TABLEV INTERFERENCES Data on retention
times obtained Retention
Ethosuximide Procainamide Procaine Primidone Lidocaine Phenobarbital Amobarbital Secobarbital Pentobarbital Propoxyphene Glutethimide Methaqualone
by cbromatographing ~__
pure standard
solutions.
time @in)
3.4 4.0 5.8 6.4 7.8 9.0 >12.0 >12.0 >12.0 >12.0 >12.0 >12.0
lidocaine and procainamide. Pure drug solutions were chromatographed individually and retention times of each noted. Drugs tested were ethosuximide, primidone, phenobarbital, amobarbital, pentobarbital, secobarbital, propoxyphene, glutethimide and methaqualone. Ethosuximide was found to elute at 3.4 min, before procainamide (4 min). Primidone eluted at 6.4 min, after procaine (5.8 min) and before lidocaine (7.8 min). Phenobarbital eluted at 9 min, after lidoCaine (7.8 min). The other drugs tested eluted after 12 min. None of the tested compounds constituted an interference under the conditions of the analysis. These results are summarized in Table V. Discussion
The value of regular measurements of serum or plasma concentrations of drugs used for treatment of cardiac arrhythmias has been well established [ 71. The therapeutic concentrations of lidocaine and procainamide pose special problems to the analyst: special columns have been recommended for gas chromatographic procedures [ 91; relatively large sample sizes; different analytical procedures are generally recommended for the individual drugs. The data presented in this report indicates that high pressure liquid chromatography using one set of operating conditions is suitable for the determination of lidoCaine and procainamide in serum. The sensitivity, precision and accuracy are satisfactory. For those samples analyzed by parallel methods, the correlation was good. Detection by absorption at 205 nm gives adequate sensitivity but poor selectivity. This lack of selectivity is largely offset by the chromatographic separation. A useful feature of the detection system is that eluted analytes may be recovered for additional investigation. The analysis time for a single sample is about 15 min, including extraction and chromatography. The use of procaine as an internal standard has advantages because of its retention time and extraction characteristics. Some sera may contain significant amounts of procaine hydrolase. Therefore sera should be extracted promptly after addition of the internal standard. In conclusion, the high pressure liquid chromatographic procedure for the
524
routine determination to other procedures.
of lidocaine
and procainamide
offers a useful alternative
References 1 2 3 4
Vesell, ES. and Passananti, Koch-Messer, J. and Klein, Atkinson, A.J. (1972) Clin. Aggarwal, V. and Bath, R. Press, Cleveland, Ohio 5 Barnhardt, F.E. (1975) in land, Ohio
G.T. (1971) Clin. Chem. 17,851 S.W. (1971) Am. Med. Assoc. 215, 1454 Chem. 18.643 (1975) in Methodology for Analytical Toxicology Methodology
for Analytical
Toxicology
(Sunshine,
(Sunshine,
I., ed.), CRC
I. ed.). CRC Press, Cleve-
6 Strong, J.M. and Atkinson, A.J. (1972) Anal. Chem. 44.2287 7 Bayes, R.N., Scott, D.B., Jebson. P.J., Goodman. M.J. and Julian, D.G. (1971) Clin. Pharmacol. Ther. 12,105 8 Meola, J. and Vanko, M. (1974) Clin. Chem. 20, 184 9 Meola, J. (1975) in Methodology for Analytical Toxicology (Sunshine, I., ed.), CRC Press, Cleveland, Ohio
