Journal of Analytical Toxicology, Vol. 16, March/April 1992
Determinationof Procaine in Equine Plasmaand Urine by High-PerformanceLiquid Chromatography A.J. Stevenson* and M.P. Weber
Race Track Division, Agriculture Canada, Ottawa and Jerseyville, Ontario F. Todi, M. Mendonca, and J.D. Fenwick Lynn & Johnston Laboratories, Lachine, Quebec
L. Young, E. Kwong, and F. Chen Can Test Ltd., Vancouver, British Columbia P. Beaumier, S. Timmings, and D. Woodard
Mann Testing Laboratories, Mississauga, Ontario S. Kacew
Department of Pharmacology, University of Ottawa ! Abstract ! The variability in plasma and urine equine procaine measurement between three independent laboratories using current methods led to the development of a sensitive, reliable, and reproducible high-performance liquid chromatographic method. Standardbred mares were administered either a penicillin G procaine preparation intramuscularly or procaine hydrochloride subcutaneously, and blood and urine were collected at defined time intervals. By HPLC the detection limits for procaine in plasma and urine were 1 and 10 ng/mL, respectively. In contrast procaine in plasma could not be detected by GC-NPD, while the urinary detection limit was 50 ng/mL. The concentration of fluoride in the collection tubes and repetitive freeze-thawing modified plasma procaine measurement. Urinary pH was a factor in estimation of urine procaine levels with greater recovery and reproducibility of results at pH 5 as compared to pH 7. This HPLC method provides a simple, sensitive, and reliable quantitation of procaine in equine plasma and urine.
samples. Because procaine--regardless of source--could affect the outcome of a race, a highly sensitive method of measurement in horse plasma and urine is necessary. Several procedures have been published for the detection and measurement of procaine in biological fluids, including spectrophotometric (5), fluorimetric (6) and thin-layer chromatographic (2,7). However, the susceptibility of procaine to hydrolysis by plasma esterases (2,8) resulted in poor recoveries and reproducibility. With the introduction of gas-liquid chromatography, a more reliable and sensitive method for measurement of procaine was developed (9). Despite this marked improvement in procaine estimation using gas chromatography, the variability in drug concentration values between our three-independent laboratories was too high and the method lacked sufficient sensitivity. The aim of this paper was to develop a more sensitive, simple, and reproducible method for the measurement of procaine by high-performance liquid chromatography (HPLC).
Experimental Introduction
The use of local anesthetics in medication of race horses is prohibited by most racing authorities. Administration of procaine hydrochloride as a local anesthetic to mask lameness in racing horses is clearly outlawed (1). In addition, the central stimulant actions of procaine hydrochloride may enhance race performance and is clearly a basis for drug prohibition (2). However, a dilemma arises when procaine is utilized for reasons unrelated to local anesthesia or central stimulation. The ability of procaine to form salts with other drugs and thus prolong pharmacological actions of these agents has been exploited therapeutically. The combination of procaine with penicillin results in a low solubility preparation, which is clinically useful to maintain the antibiotic action of penicillin (3). It should be noted that procaine is also present for a prolonged duration when procaine-penicillin is administered. Although the long-acting antibacterial actions of procaine-penicillin are desirable, the sustained presence of procaine from this preparation has created considerable problems for the racing authorities (3,4). The source of procaine cannot be determined based on analysis of post race 9 Author to whom correspondence should be addressed. Full address: A.J. Stevenson, Race Track Division, Agriculture Canada, P.O. Box 5904, Station F, Ottawa, Ontario K2C 3X7, Canada
The reference standards procaine hydrochloride and tetracaine hydrochloride were obtained from Sigma Chemical Company. All chemicals and solvents used were analytical grade or better. The drugs administered, drug sources, dosage, and routes of administration are shown in Table I. One to four standardbred mares weighing between 400 and 500 kg were administered either 200 mg of procaine hydrochloride subcutaneously or 1.656 g of penicillin procaine intramuscularly (Table I). Sodium fluoride (NaF) study
In this study, blood was collected 30-40 min after drug administration into one of the following commercially available vacuum tubes: BD 6526, no NaF; BD 6428, 2.5 mg/mL NaF; BD 6479, 4.3 mg/mL NaF; BD 3670 10 mg/mL NaF. Based on these findings blood samples in subsequent experiments were collected using 25-mL syringes (Monovette Sarstedt) into BD 3670 Vacutainer | tubes containing sodium fluoride (250 mg) to yield a final concentration of 10 mg fluoride per 1 mL of blood, which is sufficient to inhibit hydrolysis by plasma esterases, as previously described by Baselt (10,11). HPLC blood study
In this study, blood was collected from 10 to 360 min after procaine hydrochloride and 1 to 84 h after procaine penicillin. In the
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.
93
Journal of Analytical Toxicology, Vol. 16, March/April 1992
Table I. Procaine-Containing Products Used in this Study* Trade Name
Drug Penicillin G procaine
Procaine hydrochloride
Source
Procaine dose
Volume (mL)
Route of administration
Type of experiment
Azimycin
Schering Canada, Pointe Claire, Quebec
1.656 g
20
im
HPLC GC/NPD
Pen-di-Strep
Rogar/STB, London, Ontario
1.656 g
20
im
pH effects freeze/thaw effects
Cillimycin
Austin Labs. Joliette, Quebec
1.656 g
20
im
sodium fluoride effects
Novocain
Winthrop Lab., Div. of Sterling Drug, Aurora, Ontario
200 mg
5
sc
HPLC
*HPLC was used to measure procaine levels in all types of experiments including the effects of pH, freeze-thaw, and sodium fluoride.
UrneSam0es 1 IBo~ 1
Adjustedto pH 5 and Frozen
NaF10 mg/mL and Frozen
for Analysis Urine: 5 mL
|
Plasma/Blood:4 mL
l
I 3-301agadd TetracaineM I I /
Preparedin Drug
I
FreeU Plasmaor .... r1
To pH 9.5 with ~ I Urine:03-30 pg/L / BoraxBuffer _,) ~Plasma:1-250ng/mLJ
l BlOOd 1 pg Tetracame add To pH 95 with BoraxBuffer
D=chloromethane
1 I Comdmed Orgamc Ex:racts Evaporatedto Dryness UnderNdrogen at 6O'C
Residue Reconstduted in Methanol (1 0 mL) 20-100 ,uLInjected in HPLC
Residue Reconsntuted m MeIhanol (0 2-2 0 mL) 125 ~L Injected m HPLC
Figure 1. Schematicdiagramfur the analysisof procainein equineplasma and urine by HPLC. GC-NPD experiment blood was collected from 15 min to 6 h alter procaine penicillin. After collection the blood samples were immediately centrifuged, and the plasma was removed and stored at-16~ for shipment, as shown in Figure 1. To establish the stability of procaine in blood constituents, whole blood was removed, stored at -16~ and shipped for analysis.
Urine HPLC study Urine samples were obtained via an indwelling Foley catheter prior to and at 1-24 h post procaine hydrochloride administration for the HPLC experiments. In the case of procaine-penicillin, urine samples were collected prior to and at 1-48 h alter drug ad94
ministration for HPLC analysis. In the comparative study between GC-NPD and HPLC, urine was collected prior to and at either 0-3 or 3-6 h in different horses. The pH of the urine samples was adjusted to 5 before freezing. The extraction procedures of procaine from plasma and urine are shown in Figure 1. The plasma and urine extracts were reconstituted in 200 pL and 1 mL methanol, respectively, and aliquots were taken for HPLC analysis. The recovery of procaine and internal standard tetracaine from plasma and urine was approximately 90%.
Instrumental parameters The levels of procaine in plasma and urine were measured with a high-performance liquid chmmatog,aph (Waters Scientific) automated system of two Model 6000A pumps, a Model 710 WISP automatic injector, a Model 720 system controller, UV detector Model 481, and Hewlctt-Packard integrato," Model 3396. The instrument was cquipped with a Radial Pak Cis column (l()-Nn particle size). Separation was made with a mobile phase of acetnnitrilc-0.0165M triethylaminc t 85:15) adjusting pH to 3 with concentrated H~PO.~ at at llow rate of 2.0 mlJmin. The detector wavelength was fixed at 288 nm. "Fhedetection limits for procaine were 1.0 ng/mL in plasma and 10 nghnL in urine. For comparative pt, rposes plasma and urine extracts were analyzed for procaine with an HP Model 5890 gas chromatograph equipped with a nitrogen-phosphorus detecto," (NPD), as previously described (12). The instrument was equipped with a 2-m • 2-ram internal dialneter glass column packed with 3% OV-17 Chromosorb WHP 8(1/100 mesh. The detector and injector port temperatures were 300 and 250~ respectively, The oven temperatu,e was isothermal at 220~ Helium was used as a carrier gas at a flow rate of 40 mL/min. The identities of compounds were confirmed by comparing the TLC Rt, values and by gas ch,omatography/nlass spectrometry (GC/MS).
Results and D i s c u s s i o n The ability to estimate and attain reproducible values of procaine in plasma is highly dependent on the collection procedure. Figure 2 illustratcs the importance of sodium fluoride, in the collection tube on plasma procaine levels, as determined by HPLC following penicillin procaine administration. In tubes containing 0 to 4.3 nag/mL sodium fluo,ide, plasma procaine levels were in the 20-ng/mL range at Day I and vi,'tually disappeared by Day 4. In contrast, in tubes containing 5 to 10 mg/mL sodium fluoride, plasma procaine levels were between 85 and
Journal
of A n a l y t i c a l T o x i c o l o g y ,
Vol. 16, M a r c h / A p r i l
1992
110 ng/mL and remained at that level when stored for 7 days. Similarly, Tanaka and Momose (13) found that in tubes containing either 5, 10, or 20 mg/mL sodium fluoride the plasma procaine levels were maintained for 19 days after collection. In the presence of 3.6 mg/mL sodium fluoride, the procaine plasma levels fell by 70% after 5 days. Our data confirm that at the time of blood sampling the conce0tration of sodium fluoride in collection tubes is essential to prevent hydrolysis of procaine by plasma esterases. The susceptibility of procaine to hydrolysis by plasma esterases is well-known (4,7), and the ability of fluoride to inhibit these enzymes has been reported (10,11). However, this study clearly shows the minimal amount of fluoride necessary to preserve procaine plasma levels in equine samples. In order to assess whether procaine stability is affected by blood constituents, blood was collected in tubes containing 10 mg/mL of sodium fluoride from horses administered penicillin-procaine. In one case blood was maintained whole and frozen and then shipped for analysis. In the other instance whole blood was centrifuged, the plasma removed and frozen, and then shipped for analysis. Data in Figure 3 illustrate that procaine levels as measured by HPLC are maintained in whole blood after 1 to 7 days at an equivalent concentration. However, analysis of plasma showed a 50% decline in procaine values at 7 days as compared to 1 day values. It is of interest that Tobin et al. (7,14) also found significantly greater concentrations of procaine in whole blood compared to plasma. The finding that even in the presence of 10 mg/mL sodium fluoride procaine levels were maintained in whole blood but markedly declined in plasma over 7 days indicates that binding of drug to red cells protects procaine from hydrolysis by serum esterases (15). Indeed, Tobin et al. (14) demonstrated that procaine levels were significantly higher in the red cell fraction compared to plasma. In the frozen state concentrations of drugs or cellular constituents are maintained without appreciable decline. Analysis is thus carried out on freshly thawed samples. As shown in Table A
II, procaine levels were determined by HPLC in blood collected at various times from horses administered procaine hydrochloride. In comparison between once-and twice-thawed samples from 10 to 60 min, the procaine levels were approximately twice as great after one thaw. Thus there is a 50% loss of procaine when freeze-thawing is repeated twice. In samples collected from 120 to 360 min a repetitious freeze-thaw process did not affect procaine levels, as drug levels were at the limits of detection in both cases. We attempted to measure plasma procaine by GC-NPD and compare these values with those obtained by HPLC. Unfortunately, we were unable to obtain plasma procaine measurements with GC-NPD because an interfering substance coeluted with Table II. Effects of Freeze-Thawing on Plasma Procaine Levels Measured by HPLC* Procaine concentration(ng/mL)
.... '1~ ......
Thawed oncet
Thawed twice**
10 20 30 40 50 6O 120 180 240 300 360
48+15 74+12 89• 66+_11 57 + 9 47 + 7 18+4 10+3 7+-2 4+2 3•
26• 40+_8 56• 38+_9 27 +_10 28 +_7 13+4 10+4 6+-3 5+_3 3•
9 Blood w a s o b t a i n e d from horses a d m i n i s t e r e d 2 0 0 mg procaine hydrochloride s u b c u t a n e o u s l y frozen a n d t h a w e d once for analysis. T h e s a m e blood was then refrozan a n d t h a w e d again (twice) for analysis. t M e a n s + S . E . M of 8 horses. "* M e a n s + S . E M . of 6 horses.
:
pl...
A..........,,..........~::::.-...o...........................
Time (min)
B
.... 0 . . . .... ~,... .... 9 ,.-.... r .... O - "
..,,.. . . . . . . . . . . . . . .
~,.r. . . . . . .
SO i O / ' d II ~ / ~ t . 4.',, ,,,l/mr, it.U Big/IlL O am/I.
A
n
& .......... & ......................
%'...
9
.... v . . . .... &...
*V ....
L u
.~
.... " ' ~ ..................................
~... " ' " . , ~ ..... .
.....~ ........:~.:::::::::~ eo
| e9
I;0
a e,
tll
4 0.
................... e o:
e.e
.
. Yo
~'~
,
,:o
;
..o
(dayl)
0.:o
,o.:
true
(cJoye)
;
LO
;
M
9
,'0
=.'."::..::..e, ......... ~, .... ...
" o .....
"'".,s ........................
, ,,.., i& ....
it: ...........
Ooe
d,'
2,
.!,
,!,
,!,-
.",
,!,
t ltllm ( d a y 8 )
Figure 2. Plasma was collected from a horse administeredpenicillin G procaine intramuscularly into tubes containing varying amounts of sodium fluoride. (A) Influence of varying concentrations of fluoride on procaine levels expressedas ng/mL plasma; (B) effects of storage on procaine measurement at certain fluoride concentrations. Dataare expressed as a percent of Day 1 values.
..,-,~
"V.
.... o . . . t o m o / L .... & - . . II I I O / I L .... ~ . - - 4 . 3 * m l n t . .... 0 . . , | . I I N I L
"":::":'~:'"" ""'~::::: .....
~" .......... & ..............................
.... '~-.. PI.&IIMA
i'o
.!.
2o
.!. ttu
2~
,!.
2.
,!.
(doym)
Figure 3. Blood was collected from a horse administered penicillin G procaine intramuscularly into tubes containing 10 mg/mL sodium fluoride and analyzed for procaine. A portion of this blood was centrifuged and plasma obtained was analyzed for procaine. Data are shown in A expressed as ng/mL. Graph B represents the analysis of procaine in stored blood or plasma and expressed as percent of Day 1 values.
95
Journal of Analytical Toxicology, Vol. 16, March/April 1992
Table III. Influence of pH on Urinary Procaine Levels Measured by HPLC* Procaine concentration(ng/mL)
[P n o c m i n e
I~mnicillin |1
|
IProcatno
Q ~' A
'r XZ 'rXX
Lab
pH 5
pH 7
I II Ill
2200 1720 1920
860 410 780
9Urine was collectedfrom a horse administeredprocainepenicillin G, pH adjusted, frozen and aliquots shipped for analysis by threeindependentlabs. The pH was readjustedat each lab separatelyas required.
nary procaine was detected by GC-NPD, the detection sensitivity was markedly greater with HPLC.
HCl]
Acknowledgments A
~
0
z time
_t
0't)
Figure 4. Urinewas collectedfrom horsesadministeredeitherprocainehydr0chloride or penicillinG procaine. The urine was frozen and aliquots shipped to three different labs for HPLC analysis. Data are expressed as ng/mL.
procaine. In our three independent labs there was minimal interference in the region of interest using the HPLC method. Although Smith etal. (9) were successful in the measurement of plasma procaine by GC, the detection limit in plasma was 10 ng/mL. However, under our HPLC conditions the detection limit for plasma procaine was 1 ng/mL, a 10-fold increase in sensitivity. Data in Figure 4 illustrate the urinary procaine values obtained by HPLC in three independent labs in horses given either penicillin-procaine or procaine hydrochloride. Regardless of the source of procaine, there were no marked differences in the urinary drug levels between the three labs at any of the collection times confirming a reliability in methodology. The urinary elimination pattern noted in our study was similar to that previously reported (16,17). Despite the fact that horses in this study received far less procaine hydrochloride or procaine penicillin, the concentration of drug and duration of detection were equal to those reported in previous studies (16,17). This study confirmed that HPLC estimation was a more sensitive technique than GC-NPD because the detection limit for urinary procaine was l0 ng/mL for the former and 50 ng/mL for the latter. Table III shows that urinary pH plays an important role in urinary procaine determination. At pH 5 urinary procaine values measured were 2-4 fold higher than urine at pH 7. Evans and Lambert (l) also found that detection of urinary procaine was significantly reduced when pH was increased from 5 to 7.
Conclusion
The quantitative analysis of procaine in plasma and urine, regardless of whether a procaine penicillin G preparation or procaine hydrochloride was administered, was sensitive and reliable using HPLC. The concentration of procaine in plasma was (1) affected by the amount of sodium fluoride in the collection tube, (2) less than that in whole blood, and (3) reduced by repetitive freeze-thaw procedures. In comparison, plasma procaine could not be adequately quantified by GC-NPD. Although uri96
This study was funded wholly by the Race Track Division of Agriculture Canada. The authors gratefully acknowledge the assistance of the staff of the Equine Drug Evaluation Center, Jerseyville, Ontario.
References 1. J.A. Evans and M.B.T. Lambert. Estimation of procaine in urine of horses. Vet. Rec. 95:316-18 (1974). 2. T. Tobin, C.Y. Tai, and S. Arnett. Recovery o1 procaine from biological fluids. Res. Commun. Chem. PathoL PharmacoL 11:187-94 (1975). 3. T. Tobin, C.Y. Tai, J. O'Leary, L. Sturma, and S. Amett. Pharmacology of procaine in the horse: evidence against the existence of a "procaine~enicillin" complex. Am. J. Vet. Res. 38:437-42 (1977). 4. T. Tobin, J.W. Blake, C.Y. Tai, and S. Arnett. Pharmacology of procaine in the horse: a preliminary report. Am. J. Vet. Res. 37:1107-10 (1976). 5. S.C. Lynn. Studies on race horse cholinesterase. Proceedings of the 17th Meeting of the Association of Official Racing Chemists, 1962, pp. 219-28. 6. S. Udenfriend, D.E. Duggan, B.M Vesta, and B.B. Brodie. A spectrophotofluorometric study of organic compounds of pharmacologic interest. J. PharmacoL Exp. The~ 120:26-32 (1967). 7. T. Tobin, J.W. Blake, C.Y. Tai, L. Sturma, and S. Arnett. Pharmacology of procaine in the horse: procaine esterase properlies of equine plasma and synovial fluid. Am. J. Vet, Res. 37:1165-70 (1976). 8. W. Kalow. Hydrolysis of local anesthetics by human serum cholinesterase. J. PharmacoL Exp. Ther. 104:122-34 (1952). 9. R H Smith, M.A. Brewster, J.A. MacDonald, and D.S. Thompson. Measurement of chloroprocaine and procaine in plasma by flame ionization gas-liquid chromatography. Clin. Chem. 24:1599-1602 (1978). 10. R.C. Baselt, R.F. Shaw, and R. McEvilly. Effect of sodium fluoride on cholinesterase activity in postmortem blood. J. Forens. Sci. 30:1206-1209 (1985). 11. R.C. Baselt. Stability of cocaine in biological fluids. J. Chromatogr. 268: 502-505 (1983). 12. P. Beaumier, S. Timmings, J.D. Fenwick, F. Todi, L.M. Young, T. Yeow, M Weber, and A.J. Stevenson. Procaine detection after administering various procaine formulations to standardbreds. Proceedings of the 6th International Conference of Racing Analysts and Veterinarians, Hong Kong, 1985, pp. 209-16. 13. T. Tanaka and A. Momose. Inhibitory effect of neostigmine bromide and sodium fluoride on the degradation of procaine and tetracaine in equine blood. Proceeding of the 6th International Conference of Racing Analysts and Veterinarians, Kentucky, 1988, pp. 203-207. 14. T. Tobin, J.W. Blake, L. Sturma, S, Arnett, and J. Truelove. Pharmacology of procaine in the horse: pharmacokinetics and behavioural effects. Am. J. Vet. Res. 38:637-47 (1977). 15. M M Reidenberg. The procaine esterase activity of serum from different mammalian species. Proc. Soc. Exp. BioL Med. 140" 1059-61 (1972). 16. T. Tobin and J.W. Blake. A review of the pharmacology, pharmacokinetics and behavioural effects of procaine in thoroughbred horses. Brit. J. Sports Med. 10:109-16 (1976). 17. T. Tobin and J.W. Blake. The pharmacology of procaine in the horse: relationships between plasma and urinary concentrations of procaine. J. Equine Med. Surg. 1:188-94 (1977). Manuscript received October 15, 1990; revision received May 1, 1991.
Determinationof Procaine in Equine Plasmaand Urine by High-PerformanceLiquid Chromatography A.J. Stevenson* and M.P. Weber
Race Track Division, Agriculture Canada, Ottawa and Jerseyville, Ontario F. Todi, M. Mendonca, and J.D. Fenwick Lynn & Johnston Laboratories, Lachine, Quebec
L. Young, E. Kwong, and F. Chen Can Test Ltd., Vancouver, British Columbia P. Beaumier, S. Timmings, and D. Woodard
Mann Testing Laboratories, Mississauga, Ontario S. Kacew
Department of Pharmacology, University of Ottawa ! Abstract ! The variability in plasma and urine equine procaine measurement between three independent laboratories using current methods led to the development of a sensitive, reliable, and reproducible high-performance liquid chromatographic method. Standardbred mares were administered either a penicillin G procaine preparation intramuscularly or procaine hydrochloride subcutaneously, and blood and urine were collected at defined time intervals. By HPLC the detection limits for procaine in plasma and urine were 1 and 10 ng/mL, respectively. In contrast procaine in plasma could not be detected by GC-NPD, while the urinary detection limit was 50 ng/mL. The concentration of fluoride in the collection tubes and repetitive freeze-thawing modified plasma procaine measurement. Urinary pH was a factor in estimation of urine procaine levels with greater recovery and reproducibility of results at pH 5 as compared to pH 7. This HPLC method provides a simple, sensitive, and reliable quantitation of procaine in equine plasma and urine.
samples. Because procaine--regardless of source--could affect the outcome of a race, a highly sensitive method of measurement in horse plasma and urine is necessary. Several procedures have been published for the detection and measurement of procaine in biological fluids, including spectrophotometric (5), fluorimetric (6) and thin-layer chromatographic (2,7). However, the susceptibility of procaine to hydrolysis by plasma esterases (2,8) resulted in poor recoveries and reproducibility. With the introduction of gas-liquid chromatography, a more reliable and sensitive method for measurement of procaine was developed (9). Despite this marked improvement in procaine estimation using gas chromatography, the variability in drug concentration values between our three-independent laboratories was too high and the method lacked sufficient sensitivity. The aim of this paper was to develop a more sensitive, simple, and reproducible method for the measurement of procaine by high-performance liquid chromatography (HPLC).
Experimental Introduction
The use of local anesthetics in medication of race horses is prohibited by most racing authorities. Administration of procaine hydrochloride as a local anesthetic to mask lameness in racing horses is clearly outlawed (1). In addition, the central stimulant actions of procaine hydrochloride may enhance race performance and is clearly a basis for drug prohibition (2). However, a dilemma arises when procaine is utilized for reasons unrelated to local anesthesia or central stimulation. The ability of procaine to form salts with other drugs and thus prolong pharmacological actions of these agents has been exploited therapeutically. The combination of procaine with penicillin results in a low solubility preparation, which is clinically useful to maintain the antibiotic action of penicillin (3). It should be noted that procaine is also present for a prolonged duration when procaine-penicillin is administered. Although the long-acting antibacterial actions of procaine-penicillin are desirable, the sustained presence of procaine from this preparation has created considerable problems for the racing authorities (3,4). The source of procaine cannot be determined based on analysis of post race 9 Author to whom correspondence should be addressed. Full address: A.J. Stevenson, Race Track Division, Agriculture Canada, P.O. Box 5904, Station F, Ottawa, Ontario K2C 3X7, Canada
The reference standards procaine hydrochloride and tetracaine hydrochloride were obtained from Sigma Chemical Company. All chemicals and solvents used were analytical grade or better. The drugs administered, drug sources, dosage, and routes of administration are shown in Table I. One to four standardbred mares weighing between 400 and 500 kg were administered either 200 mg of procaine hydrochloride subcutaneously or 1.656 g of penicillin procaine intramuscularly (Table I). Sodium fluoride (NaF) study
In this study, blood was collected 30-40 min after drug administration into one of the following commercially available vacuum tubes: BD 6526, no NaF; BD 6428, 2.5 mg/mL NaF; BD 6479, 4.3 mg/mL NaF; BD 3670 10 mg/mL NaF. Based on these findings blood samples in subsequent experiments were collected using 25-mL syringes (Monovette Sarstedt) into BD 3670 Vacutainer | tubes containing sodium fluoride (250 mg) to yield a final concentration of 10 mg fluoride per 1 mL of blood, which is sufficient to inhibit hydrolysis by plasma esterases, as previously described by Baselt (10,11). HPLC blood study
In this study, blood was collected from 10 to 360 min after procaine hydrochloride and 1 to 84 h after procaine penicillin. In the
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.
93
Journal of Analytical Toxicology, Vol. 16, March/April 1992
Table I. Procaine-Containing Products Used in this Study* Trade Name
Drug Penicillin G procaine
Procaine hydrochloride
Source
Procaine dose
Volume (mL)
Route of administration
Type of experiment
Azimycin
Schering Canada, Pointe Claire, Quebec
1.656 g
20
im
HPLC GC/NPD
Pen-di-Strep
Rogar/STB, London, Ontario
1.656 g
20
im
pH effects freeze/thaw effects
Cillimycin
Austin Labs. Joliette, Quebec
1.656 g
20
im
sodium fluoride effects
Novocain
Winthrop Lab., Div. of Sterling Drug, Aurora, Ontario
200 mg
5
sc
HPLC
*HPLC was used to measure procaine levels in all types of experiments including the effects of pH, freeze-thaw, and sodium fluoride.
UrneSam0es 1 IBo~ 1
Adjustedto pH 5 and Frozen
NaF10 mg/mL and Frozen
for Analysis Urine: 5 mL
|
Plasma/Blood:4 mL
l
I 3-301agadd TetracaineM I I /
Preparedin Drug
I
FreeU Plasmaor .... r1
To pH 9.5 with ~ I Urine:03-30 pg/L / BoraxBuffer _,) ~Plasma:1-250ng/mLJ
l BlOOd 1 pg Tetracame add To pH 95 with BoraxBuffer
D=chloromethane
1 I Comdmed Orgamc Ex:racts Evaporatedto Dryness UnderNdrogen at 6O'C
Residue Reconstduted in Methanol (1 0 mL) 20-100 ,uLInjected in HPLC
Residue Reconsntuted m MeIhanol (0 2-2 0 mL) 125 ~L Injected m HPLC
Figure 1. Schematicdiagramfur the analysisof procainein equineplasma and urine by HPLC. GC-NPD experiment blood was collected from 15 min to 6 h alter procaine penicillin. After collection the blood samples were immediately centrifuged, and the plasma was removed and stored at-16~ for shipment, as shown in Figure 1. To establish the stability of procaine in blood constituents, whole blood was removed, stored at -16~ and shipped for analysis.
Urine HPLC study Urine samples were obtained via an indwelling Foley catheter prior to and at 1-24 h post procaine hydrochloride administration for the HPLC experiments. In the case of procaine-penicillin, urine samples were collected prior to and at 1-48 h alter drug ad94
ministration for HPLC analysis. In the comparative study between GC-NPD and HPLC, urine was collected prior to and at either 0-3 or 3-6 h in different horses. The pH of the urine samples was adjusted to 5 before freezing. The extraction procedures of procaine from plasma and urine are shown in Figure 1. The plasma and urine extracts were reconstituted in 200 pL and 1 mL methanol, respectively, and aliquots were taken for HPLC analysis. The recovery of procaine and internal standard tetracaine from plasma and urine was approximately 90%.
Instrumental parameters The levels of procaine in plasma and urine were measured with a high-performance liquid chmmatog,aph (Waters Scientific) automated system of two Model 6000A pumps, a Model 710 WISP automatic injector, a Model 720 system controller, UV detector Model 481, and Hewlctt-Packard integrato," Model 3396. The instrument was cquipped with a Radial Pak Cis column (l()-Nn particle size). Separation was made with a mobile phase of acetnnitrilc-0.0165M triethylaminc t 85:15) adjusting pH to 3 with concentrated H~PO.~ at at llow rate of 2.0 mlJmin. The detector wavelength was fixed at 288 nm. "Fhedetection limits for procaine were 1.0 ng/mL in plasma and 10 nghnL in urine. For comparative pt, rposes plasma and urine extracts were analyzed for procaine with an HP Model 5890 gas chromatograph equipped with a nitrogen-phosphorus detecto," (NPD), as previously described (12). The instrument was equipped with a 2-m • 2-ram internal dialneter glass column packed with 3% OV-17 Chromosorb WHP 8(1/100 mesh. The detector and injector port temperatures were 300 and 250~ respectively, The oven temperatu,e was isothermal at 220~ Helium was used as a carrier gas at a flow rate of 40 mL/min. The identities of compounds were confirmed by comparing the TLC Rt, values and by gas ch,omatography/nlass spectrometry (GC/MS).
Results and D i s c u s s i o n The ability to estimate and attain reproducible values of procaine in plasma is highly dependent on the collection procedure. Figure 2 illustratcs the importance of sodium fluoride, in the collection tube on plasma procaine levels, as determined by HPLC following penicillin procaine administration. In tubes containing 0 to 4.3 nag/mL sodium fluo,ide, plasma procaine levels were in the 20-ng/mL range at Day I and vi,'tually disappeared by Day 4. In contrast, in tubes containing 5 to 10 mg/mL sodium fluoride, plasma procaine levels were between 85 and
Journal
of A n a l y t i c a l T o x i c o l o g y ,
Vol. 16, M a r c h / A p r i l
1992
110 ng/mL and remained at that level when stored for 7 days. Similarly, Tanaka and Momose (13) found that in tubes containing either 5, 10, or 20 mg/mL sodium fluoride the plasma procaine levels were maintained for 19 days after collection. In the presence of 3.6 mg/mL sodium fluoride, the procaine plasma levels fell by 70% after 5 days. Our data confirm that at the time of blood sampling the conce0tration of sodium fluoride in collection tubes is essential to prevent hydrolysis of procaine by plasma esterases. The susceptibility of procaine to hydrolysis by plasma esterases is well-known (4,7), and the ability of fluoride to inhibit these enzymes has been reported (10,11). However, this study clearly shows the minimal amount of fluoride necessary to preserve procaine plasma levels in equine samples. In order to assess whether procaine stability is affected by blood constituents, blood was collected in tubes containing 10 mg/mL of sodium fluoride from horses administered penicillin-procaine. In one case blood was maintained whole and frozen and then shipped for analysis. In the other instance whole blood was centrifuged, the plasma removed and frozen, and then shipped for analysis. Data in Figure 3 illustrate that procaine levels as measured by HPLC are maintained in whole blood after 1 to 7 days at an equivalent concentration. However, analysis of plasma showed a 50% decline in procaine values at 7 days as compared to 1 day values. It is of interest that Tobin et al. (7,14) also found significantly greater concentrations of procaine in whole blood compared to plasma. The finding that even in the presence of 10 mg/mL sodium fluoride procaine levels were maintained in whole blood but markedly declined in plasma over 7 days indicates that binding of drug to red cells protects procaine from hydrolysis by serum esterases (15). Indeed, Tobin et al. (14) demonstrated that procaine levels were significantly higher in the red cell fraction compared to plasma. In the frozen state concentrations of drugs or cellular constituents are maintained without appreciable decline. Analysis is thus carried out on freshly thawed samples. As shown in Table A
II, procaine levels were determined by HPLC in blood collected at various times from horses administered procaine hydrochloride. In comparison between once-and twice-thawed samples from 10 to 60 min, the procaine levels were approximately twice as great after one thaw. Thus there is a 50% loss of procaine when freeze-thawing is repeated twice. In samples collected from 120 to 360 min a repetitious freeze-thaw process did not affect procaine levels, as drug levels were at the limits of detection in both cases. We attempted to measure plasma procaine by GC-NPD and compare these values with those obtained by HPLC. Unfortunately, we were unable to obtain plasma procaine measurements with GC-NPD because an interfering substance coeluted with Table II. Effects of Freeze-Thawing on Plasma Procaine Levels Measured by HPLC* Procaine concentration(ng/mL)
.... '1~ ......
Thawed oncet
Thawed twice**
10 20 30 40 50 6O 120 180 240 300 360
48+15 74+12 89• 66+_11 57 + 9 47 + 7 18+4 10+3 7+-2 4+2 3•
26• 40+_8 56• 38+_9 27 +_10 28 +_7 13+4 10+4 6+-3 5+_3 3•
9 Blood w a s o b t a i n e d from horses a d m i n i s t e r e d 2 0 0 mg procaine hydrochloride s u b c u t a n e o u s l y frozen a n d t h a w e d once for analysis. T h e s a m e blood was then refrozan a n d t h a w e d again (twice) for analysis. t M e a n s + S . E . M of 8 horses. "* M e a n s + S . E M . of 6 horses.
:
pl...
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Figure 2. Plasma was collected from a horse administeredpenicillin G procaine intramuscularly into tubes containing varying amounts of sodium fluoride. (A) Influence of varying concentrations of fluoride on procaine levels expressedas ng/mL plasma; (B) effects of storage on procaine measurement at certain fluoride concentrations. Dataare expressed as a percent of Day 1 values.
..,-,~
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.... o . . . t o m o / L .... & - . . II I I O / I L .... ~ . - - 4 . 3 * m l n t . .... 0 . . , | . I I N I L
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Figure 3. Blood was collected from a horse administered penicillin G procaine intramuscularly into tubes containing 10 mg/mL sodium fluoride and analyzed for procaine. A portion of this blood was centrifuged and plasma obtained was analyzed for procaine. Data are shown in A expressed as ng/mL. Graph B represents the analysis of procaine in stored blood or plasma and expressed as percent of Day 1 values.
95
Journal of Analytical Toxicology, Vol. 16, March/April 1992
Table III. Influence of pH on Urinary Procaine Levels Measured by HPLC* Procaine concentration(ng/mL)
[P n o c m i n e
I~mnicillin |1
|
IProcatno
Q ~' A
'r XZ 'rXX
Lab
pH 5
pH 7
I II Ill
2200 1720 1920
860 410 780
9Urine was collectedfrom a horse administeredprocainepenicillin G, pH adjusted, frozen and aliquots shipped for analysis by threeindependentlabs. The pH was readjustedat each lab separatelyas required.
nary procaine was detected by GC-NPD, the detection sensitivity was markedly greater with HPLC.
HCl]
Acknowledgments A
~
0
z time
_t
0't)
Figure 4. Urinewas collectedfrom horsesadministeredeitherprocainehydr0chloride or penicillinG procaine. The urine was frozen and aliquots shipped to three different labs for HPLC analysis. Data are expressed as ng/mL.
procaine. In our three independent labs there was minimal interference in the region of interest using the HPLC method. Although Smith etal. (9) were successful in the measurement of plasma procaine by GC, the detection limit in plasma was 10 ng/mL. However, under our HPLC conditions the detection limit for plasma procaine was 1 ng/mL, a 10-fold increase in sensitivity. Data in Figure 4 illustrate the urinary procaine values obtained by HPLC in three independent labs in horses given either penicillin-procaine or procaine hydrochloride. Regardless of the source of procaine, there were no marked differences in the urinary drug levels between the three labs at any of the collection times confirming a reliability in methodology. The urinary elimination pattern noted in our study was similar to that previously reported (16,17). Despite the fact that horses in this study received far less procaine hydrochloride or procaine penicillin, the concentration of drug and duration of detection were equal to those reported in previous studies (16,17). This study confirmed that HPLC estimation was a more sensitive technique than GC-NPD because the detection limit for urinary procaine was l0 ng/mL for the former and 50 ng/mL for the latter. Table III shows that urinary pH plays an important role in urinary procaine determination. At pH 5 urinary procaine values measured were 2-4 fold higher than urine at pH 7. Evans and Lambert (l) also found that detection of urinary procaine was significantly reduced when pH was increased from 5 to 7.
Conclusion
The quantitative analysis of procaine in plasma and urine, regardless of whether a procaine penicillin G preparation or procaine hydrochloride was administered, was sensitive and reliable using HPLC. The concentration of procaine in plasma was (1) affected by the amount of sodium fluoride in the collection tube, (2) less than that in whole blood, and (3) reduced by repetitive freeze-thaw procedures. In comparison, plasma procaine could not be adequately quantified by GC-NPD. Although uri96
This study was funded wholly by the Race Track Division of Agriculture Canada. The authors gratefully acknowledge the assistance of the staff of the Equine Drug Evaluation Center, Jerseyville, Ontario.
References 1. J.A. Evans and M.B.T. Lambert. Estimation of procaine in urine of horses. Vet. Rec. 95:316-18 (1974). 2. T. Tobin, C.Y. Tai, and S. Arnett. Recovery o1 procaine from biological fluids. Res. Commun. Chem. PathoL PharmacoL 11:187-94 (1975). 3. T. Tobin, C.Y. Tai, J. O'Leary, L. Sturma, and S. Amett. Pharmacology of procaine in the horse: evidence against the existence of a "procaine~enicillin" complex. Am. J. Vet. Res. 38:437-42 (1977). 4. T. Tobin, J.W. Blake, C.Y. Tai, and S. Arnett. Pharmacology of procaine in the horse: a preliminary report. Am. J. Vet. Res. 37:1107-10 (1976). 5. S.C. Lynn. Studies on race horse cholinesterase. Proceedings of the 17th Meeting of the Association of Official Racing Chemists, 1962, pp. 219-28. 6. S. Udenfriend, D.E. Duggan, B.M Vesta, and B.B. Brodie. A spectrophotofluorometric study of organic compounds of pharmacologic interest. J. PharmacoL Exp. The~ 120:26-32 (1967). 7. T. Tobin, J.W. Blake, C.Y. Tai, L. Sturma, and S. Arnett. Pharmacology of procaine in the horse: procaine esterase properlies of equine plasma and synovial fluid. Am. J. Vet, Res. 37:1165-70 (1976). 8. W. Kalow. Hydrolysis of local anesthetics by human serum cholinesterase. J. PharmacoL Exp. Ther. 104:122-34 (1952). 9. R H Smith, M.A. Brewster, J.A. MacDonald, and D.S. Thompson. Measurement of chloroprocaine and procaine in plasma by flame ionization gas-liquid chromatography. Clin. Chem. 24:1599-1602 (1978). 10. R.C. Baselt, R.F. Shaw, and R. McEvilly. Effect of sodium fluoride on cholinesterase activity in postmortem blood. J. Forens. Sci. 30:1206-1209 (1985). 11. R.C. Baselt. Stability of cocaine in biological fluids. J. Chromatogr. 268: 502-505 (1983). 12. P. Beaumier, S. Timmings, J.D. Fenwick, F. Todi, L.M. Young, T. Yeow, M Weber, and A.J. Stevenson. Procaine detection after administering various procaine formulations to standardbreds. Proceedings of the 6th International Conference of Racing Analysts and Veterinarians, Hong Kong, 1985, pp. 209-16. 13. T. Tanaka and A. Momose. Inhibitory effect of neostigmine bromide and sodium fluoride on the degradation of procaine and tetracaine in equine blood. Proceeding of the 6th International Conference of Racing Analysts and Veterinarians, Kentucky, 1988, pp. 203-207. 14. T. Tobin, J.W. Blake, L. Sturma, S, Arnett, and J. Truelove. Pharmacology of procaine in the horse: pharmacokinetics and behavioural effects. Am. J. Vet. Res. 38:637-47 (1977). 15. M M Reidenberg. The procaine esterase activity of serum from different mammalian species. Proc. Soc. Exp. BioL Med. 140" 1059-61 (1972). 16. T. Tobin and J.W. Blake. A review of the pharmacology, pharmacokinetics and behavioural effects of procaine in thoroughbred horses. Brit. J. Sports Med. 10:109-16 (1976). 17. T. Tobin and J.W. Blake. The pharmacology of procaine in the horse: relationships between plasma and urinary concentrations of procaine. J. Equine Med. Surg. 1:188-94 (1977). Manuscript received October 15, 1990; revision received May 1, 1991.
