- Email: [email protected]
Use of Unopettes for salicylate determinations
MICROCHEMICAL
JOURNAL
16, 268-272 (1971)
Use of Unopettes
for
RUTH La Rabida
Salicylate
Determinations
E. HEINEN*
Jackson Park Sanitarium, Chicago, Illinois AND
HORACE W. GERARDE Department of Biochemistry, School of Dentistry, Fairleigh Dickinson University, Teaneck, New Jersey 07666 Received
December
12, 1970
The determination of blood salicylate levels is required to assessthe level of medication, where intensive salicylate therapy is indicated (i.e., rheumatic fever) and in casesof toxicity due to purposeful or accidental ingestion of salicylates (i.e., aspirin, oil of wintergreen). A simple, accurate method for rapid evaluation of salicylate levels is needed, especially when intoxication is indicated. The determination of salicylates, by the use of Trinder’s reagent (I ) , was adopted by our laboratory in March, 1968. One hundred to 150 salicylate determinations are performed monthly to assessaspirin therapy for rheumatic fever patients. This afforded us a method, which both precipitated proteins and developed a purple color when salicylates reacted with ferric salts. A serum blank, as required by methods not precipitating proteins was, therefore, obviated. In addition, the color formed is stable up to 1 hour, while the method previously employed was to be read at 1 minute after the serum addition. We can now run all of our determinations concomitantly, rather than singly. The Unopette (R) is well established as a useful, accurate, disposable device for use in the hematology laboratory (2), which is now finding increasing use in the clinical chemistry and toxicology laboratory (3). Trinder’s method for the salicylate determination has been incorporated into the Unopette System. The Unopette consists of a reservoir containing 2.5 ml of Trinder’s reagent, pressure filters, and salicylate Uno* Present address: Mrs. Ruth E. Heinen, B.S., MT (ASCP), Tempe Community Hospital (Laboratory), 1.500 S. Mill Avenue, Tempe, Arizona 8528 1. 268
DETERMINATION
OF SALlCYLATES
269
l.BLOOD, SERUM, PLASMA, SPINAL FLUID, URINE.
&HAKE
VIGOROUSLY
TRINDER’S REAGENT 2.5 ml
4.
MEASURE ABSORBANCE BETWEEN 15.30 MINUTES AT 540 nm AGAINST REAGENT BLANK SET AT ZERO ABSORBANCE.
5.CALCULATlONS: SALICYLATES
hg/lOO
ml) =
FIG. 1. Salicylate in biological fluids.
paks of 100 pg, which, when diluted, yield a standard of 20 mg%, Fig. 1. To determine salicylate levels, 0.5 ml of serum, urine, whole blood, or spinal fluid are added to the reservoir containing Trinder’s reagent. The reservoirs are shaken vigorously to precipitate all of the protein. The reservoir is converted to a centrifuge tube by telescoping Unopette pipette shields into the reservoir, Fig. 2. After 2-3 minutes, the reservoir is spun at 2000 rpm for 5 minutes. A pressure filter is fitted into the reservoir opening and the contents are filtered into a cuvette. A standard (20 mg % ) is prepared by rinsing a Unopak containing 100 pg of salicylate into an Unopette reservoir containing 0.5 ml of water. The 0.5 ml of standard is added to 2.5 ml of Trinder’s reagent in a Unopette reservoir. This is accomplished by fitting a Unopette capillary onto the reservoir containing the standard and squeezing the solution out of the capillary into the Trinder’s solution as shown in Fig. 3. The standard and the unknowns are read against a reagent blank at 540 nm. (R) Becton, Dickinson and Co., Rutherford, N. J.
OD (unknown) __-~ OD (standard) X 20 mg 76 = mg yfi of salicylate. We have found excellent correlation of the Unopette method with the method described by Trinder, Table 1. We prefer the Unopette method
270
HEINEN AND GERARDE
FIG. 2. Unopette reservoir used as a centrifuge tube. Two telescoped Unopette capillary shields serve as a handle for the Unopette reservoir.
for a variety of reasons. All of the reagents which are stable for 2 years at room temperature come premeasured in disposable, nonbreakable containers. The only chance for error is in the measurement of the serum. There is no chance for breakage of the reservoir during centrif-
1.
SALICYLATE 100 PrJ
UNOPAK
H2O 0.5 ml
TRINDER’S REAGENT 2.5 ml
FIG. 3. Preparation of 20 mg/lOO ml salicylate standard.
DETERMINATION
271
OF SALICYLATES
TABLE 1 BLEND
Patient
CM ON JP MC ON AD RR LL RH RR MW MH JS CW KD RJ SB CW PS GM RJ CW DM MW RJ
SALICYLATE VALUES OBTAINED WITH TRINDER’S AND THE UNOPE-E PRCKEDURE (MG SALICYLATE/~OO ML BLOOD) Trinder’s
16.8 13.3 4.1 20.8 14.6 4.4 23.1 32.5 21.5 25.1 23.6 12.8 20.3 23.1 9.0 15.6 16.8 15.1 23.4 1.8 21.6 20.7 35.6 32.5 17.9
Unopette
Patient
16.6 13.6 3.9 20.9 15.0 4.9 23.6 33.0 21.8 26.0 24.0 13.2 20.9 23.6 9.2 16.1 17.5 15.7 23.5 2.0 21.6 20.1 36.5 32.3
AL PP MG ON LL GB PP GB RJ JP TS ss JW DD LL MC FG JH cs SB JH WH MR RL
METHOD
Trinder’s
16.2 26.0 13.4 2.8 20.4 23.8 33.6 21.6 21.2 18.1
2.1 30.6 16.3 22.8 22.0 18.2 14.6 11.6 13.2 22.8 14.0 3.6 8.0 21.2
Unopette
16 9 28.6 14.0 2.6 21.2 24.0 33.1 20.9 21.6 18.2 2.7 30.5 16.8 23.1 22.5 18.0
14.9 11.7 12.8 23.2 14.2 3.7 8.1 21.2
18.1
ugation, nor is there any need for cleaning glassware. If a suction cuvette is utilized, as in our laboratory, only one cuvette is involved. We have also found that in glass tubes a thin layer of precipitate remains at the top of the centifugate, even after prolonged spinning. When the centrifugate is pipetted off, invariably some of the precipitate is drawn in with the solution, yielding a falsely elevated value. Filtration of the solution without centrifugation is time consuming and demands extra glassware. Filtration of the centrifugates, while more rapid, again requires additional glassware. The pressure filter supplied with the Unopette System yields a clear solution, which will give an accurate reading and requires less time to use than transferring the centrifugate with a pipette. An important advantage and convenience of the Unopette method is the preparation of the Salicylate standard. All that is required is the rinsing of the stable standard out of the Unopak into 0.5 ml of water previously measured in a Unopette reservoir and transferring this
272
HEINEN
AND
GERARDE
to the Trinder’s reagent in a second Unopette reservoir. Finally, the amount of serum required, 0.5 ml, is small, making the method highly useful for pediatric institutions. SUMMARY The Unopette method for the determination of salicylate in blood was evaluated in our laboratory. Excellent correlation was found with the results obtained using Trinder’s reagent. The principal advantages of the Unopette procedure are accuracy and the convenience of having all of the test reagents, including the standard, pre-measured. Additional advantages are the short time needed to perform the test, and complete disposability of all of the components of the Unopette kit. REFERENCES 1. Trinder, P., Rapid determination of salicylate in biological fluids. Biochem. J. 57, 301-303 (1954). 2. Lynch, M. J., Raphael, S. S., Mellor, L. D., Spare, P. D., Hills, P., and Inwood, M. J. H., “Medical Laboratory Technology,” W. B. Saunders Co., Philadelphia, Pa. 1963. 3. Gerarde, H. W., The Unopette System in the clinical laboratory. Microchem. J. 9, 340-351 (1965).
JOURNAL
16, 268-272 (1971)
Use of Unopettes
for
RUTH La Rabida
Salicylate
Determinations
E. HEINEN*
Jackson Park Sanitarium, Chicago, Illinois AND
HORACE W. GERARDE Department of Biochemistry, School of Dentistry, Fairleigh Dickinson University, Teaneck, New Jersey 07666 Received
December
12, 1970
The determination of blood salicylate levels is required to assessthe level of medication, where intensive salicylate therapy is indicated (i.e., rheumatic fever) and in casesof toxicity due to purposeful or accidental ingestion of salicylates (i.e., aspirin, oil of wintergreen). A simple, accurate method for rapid evaluation of salicylate levels is needed, especially when intoxication is indicated. The determination of salicylates, by the use of Trinder’s reagent (I ) , was adopted by our laboratory in March, 1968. One hundred to 150 salicylate determinations are performed monthly to assessaspirin therapy for rheumatic fever patients. This afforded us a method, which both precipitated proteins and developed a purple color when salicylates reacted with ferric salts. A serum blank, as required by methods not precipitating proteins was, therefore, obviated. In addition, the color formed is stable up to 1 hour, while the method previously employed was to be read at 1 minute after the serum addition. We can now run all of our determinations concomitantly, rather than singly. The Unopette (R) is well established as a useful, accurate, disposable device for use in the hematology laboratory (2), which is now finding increasing use in the clinical chemistry and toxicology laboratory (3). Trinder’s method for the salicylate determination has been incorporated into the Unopette System. The Unopette consists of a reservoir containing 2.5 ml of Trinder’s reagent, pressure filters, and salicylate Uno* Present address: Mrs. Ruth E. Heinen, B.S., MT (ASCP), Tempe Community Hospital (Laboratory), 1.500 S. Mill Avenue, Tempe, Arizona 8528 1. 268
DETERMINATION
OF SALlCYLATES
269
l.BLOOD, SERUM, PLASMA, SPINAL FLUID, URINE.
&HAKE
VIGOROUSLY
TRINDER’S REAGENT 2.5 ml
4.
MEASURE ABSORBANCE BETWEEN 15.30 MINUTES AT 540 nm AGAINST REAGENT BLANK SET AT ZERO ABSORBANCE.
5.CALCULATlONS: SALICYLATES
hg/lOO
ml) =
FIG. 1. Salicylate in biological fluids.
paks of 100 pg, which, when diluted, yield a standard of 20 mg%, Fig. 1. To determine salicylate levels, 0.5 ml of serum, urine, whole blood, or spinal fluid are added to the reservoir containing Trinder’s reagent. The reservoirs are shaken vigorously to precipitate all of the protein. The reservoir is converted to a centrifuge tube by telescoping Unopette pipette shields into the reservoir, Fig. 2. After 2-3 minutes, the reservoir is spun at 2000 rpm for 5 minutes. A pressure filter is fitted into the reservoir opening and the contents are filtered into a cuvette. A standard (20 mg % ) is prepared by rinsing a Unopak containing 100 pg of salicylate into an Unopette reservoir containing 0.5 ml of water. The 0.5 ml of standard is added to 2.5 ml of Trinder’s reagent in a Unopette reservoir. This is accomplished by fitting a Unopette capillary onto the reservoir containing the standard and squeezing the solution out of the capillary into the Trinder’s solution as shown in Fig. 3. The standard and the unknowns are read against a reagent blank at 540 nm. (R) Becton, Dickinson and Co., Rutherford, N. J.
OD (unknown) __-~ OD (standard) X 20 mg 76 = mg yfi of salicylate. We have found excellent correlation of the Unopette method with the method described by Trinder, Table 1. We prefer the Unopette method
270
HEINEN AND GERARDE
FIG. 2. Unopette reservoir used as a centrifuge tube. Two telescoped Unopette capillary shields serve as a handle for the Unopette reservoir.
for a variety of reasons. All of the reagents which are stable for 2 years at room temperature come premeasured in disposable, nonbreakable containers. The only chance for error is in the measurement of the serum. There is no chance for breakage of the reservoir during centrif-
1.
SALICYLATE 100 PrJ
UNOPAK
H2O 0.5 ml
TRINDER’S REAGENT 2.5 ml
FIG. 3. Preparation of 20 mg/lOO ml salicylate standard.
DETERMINATION
271
OF SALICYLATES
TABLE 1 BLEND
Patient
CM ON JP MC ON AD RR LL RH RR MW MH JS CW KD RJ SB CW PS GM RJ CW DM MW RJ
SALICYLATE VALUES OBTAINED WITH TRINDER’S AND THE UNOPE-E PRCKEDURE (MG SALICYLATE/~OO ML BLOOD) Trinder’s
16.8 13.3 4.1 20.8 14.6 4.4 23.1 32.5 21.5 25.1 23.6 12.8 20.3 23.1 9.0 15.6 16.8 15.1 23.4 1.8 21.6 20.7 35.6 32.5 17.9
Unopette
Patient
16.6 13.6 3.9 20.9 15.0 4.9 23.6 33.0 21.8 26.0 24.0 13.2 20.9 23.6 9.2 16.1 17.5 15.7 23.5 2.0 21.6 20.1 36.5 32.3
AL PP MG ON LL GB PP GB RJ JP TS ss JW DD LL MC FG JH cs SB JH WH MR RL
METHOD
Trinder’s
16.2 26.0 13.4 2.8 20.4 23.8 33.6 21.6 21.2 18.1
2.1 30.6 16.3 22.8 22.0 18.2 14.6 11.6 13.2 22.8 14.0 3.6 8.0 21.2
Unopette
16 9 28.6 14.0 2.6 21.2 24.0 33.1 20.9 21.6 18.2 2.7 30.5 16.8 23.1 22.5 18.0
14.9 11.7 12.8 23.2 14.2 3.7 8.1 21.2
18.1
ugation, nor is there any need for cleaning glassware. If a suction cuvette is utilized, as in our laboratory, only one cuvette is involved. We have also found that in glass tubes a thin layer of precipitate remains at the top of the centifugate, even after prolonged spinning. When the centrifugate is pipetted off, invariably some of the precipitate is drawn in with the solution, yielding a falsely elevated value. Filtration of the solution without centrifugation is time consuming and demands extra glassware. Filtration of the centrifugates, while more rapid, again requires additional glassware. The pressure filter supplied with the Unopette System yields a clear solution, which will give an accurate reading and requires less time to use than transferring the centrifugate with a pipette. An important advantage and convenience of the Unopette method is the preparation of the Salicylate standard. All that is required is the rinsing of the stable standard out of the Unopak into 0.5 ml of water previously measured in a Unopette reservoir and transferring this
272
HEINEN
AND
GERARDE
to the Trinder’s reagent in a second Unopette reservoir. Finally, the amount of serum required, 0.5 ml, is small, making the method highly useful for pediatric institutions. SUMMARY The Unopette method for the determination of salicylate in blood was evaluated in our laboratory. Excellent correlation was found with the results obtained using Trinder’s reagent. The principal advantages of the Unopette procedure are accuracy and the convenience of having all of the test reagents, including the standard, pre-measured. Additional advantages are the short time needed to perform the test, and complete disposability of all of the components of the Unopette kit. REFERENCES 1. Trinder, P., Rapid determination of salicylate in biological fluids. Biochem. J. 57, 301-303 (1954). 2. Lynch, M. J., Raphael, S. S., Mellor, L. D., Spare, P. D., Hills, P., and Inwood, M. J. H., “Medical Laboratory Technology,” W. B. Saunders Co., Philadelphia, Pa. 1963. 3. Gerarde, H. W., The Unopette System in the clinical laboratory. Microchem. J. 9, 340-351 (1965).