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The estimation of chloroquine in blood serum
75 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE. Vol. 60. N o . 1. 1966.
THE ESTIMATION OF CHLOROQUINE IN BLOOD SERUM BY
E. M. ENSOR* Formerly Officer-in-Charge, Research Wing, Far East Land Forces
In late 1962, cases of malaria in British and Commonwealth troops, who had been infected during jungle operations in southern Thailand and whose infections did not respond to the usual therapeutic course of chloroquine, began to reach British military hospitals in the Far East. Faced with this situation, it became necessary to estimate the blood chloroquine levels attained with the standard dosage, to determine if resistance to the drug was in fact present. Samples of serum from cases and controls were sent to the Central Pathological Laboratory, Singapore, and attempts were made to estimate the chloroquine in these according to the method described by BRODm et al. (1947). It was soon found that this method was giving very low values of chloroquine compared with what were known to be present, and the Research Wing, Far East Land Forces, was given the task of working out a reliable technique. A modification of the original method was described by Dr. Richard L. Jacobs of the Department of Health, Education and Welfare, Public Health Service, Bethesda, 14, Md., U.S.A., in a personal communication. In this method the chloroquine in serum is extracted into heptane with 5 N NaOH. The heptane is washed with 0.1 N NaOH and the chloroquine re-extracted into water with 0.1 N HC1. This is then made alkaline with ammonia, and the fluorescence due to the chloroquine is read in an Aminco-Bowrnan spectrophotofluorometer. The fluorescence from an unknown sample is compared with that given by a standard made by adding a known quantity of chloroquine to normal sel'unl.
A few modifications were made (mainly in increasing the time of the initial extraction to 45 minutes from the 30 originally recommended), and the technique finally evolved is as follows: a. A mechanical shaker is used which will take 9 fiat 4-oz. medicine bottles. These are loaded: (1) 2 with 5 ml. of normal serum (serum blanks); (2) 2 with 5 ml. samples of normal serum to which 0.4 ~g. of chloroqnine base per ml. has been added (known samples); (3) Each of the remaining 5 bottles with 3 to 5 ml. (3 is the minimum) of a serum specimen to be estimated (unknown samples). *The author's thanks are due to Professor Robert C. Y. Lin for his kind permission to use the spectrophotofluorometer and other laboratory facilities in the Department of Pharmacology, University of Singapore.
76
THE ESTIMATION OF CHLOROQUINE IN BLOOD SERUM
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To each bottle is added 20 ml. of heptane, containing 1-5 ml. of isoamyl alcohol per 100 ml., and 1 ml. of 5 N NaOH. The bottles are then shaken for 45 minutes and the phases allowed to separate. The aqueous phase is discarded and 5 ml. of 0.1 N NaOH is added. The bottles are shaken for 10 minutes and the phases allowed to separate as before. As for c. above.
78 e.
£
THE ESTIMATION OF CHLOROQUINE I N BLOOD SERUM
15 ml. of the heptane phase are removed from each bottle and the remainder, with the aqueous phase, is discarded. The heptane is returned to the bottle and an amount of 0.1 N HC1 equal to the original volume of serum is added. The bottles are then shaken for 30 minutes. The heptane phase is removed by aspiration and discarded. 2 ml. of the aqueous phase is placed in the cuvette of the Aminco-Bowman spectrophotofluorometer and 1 ml. of normal NHsOH added and mixed by inversion. The samples are read, by setting the activating wavelength at 330 millimicrons and the fluorescence wave length at 390. The sensitivity control is kept at 40 throughout. Calculations
a.
b.
Percentage transmission x photomultiplier factor = relative fluorescence (RF). RF of samples, known or unknown, minus mean RF of serum blanks -=--corrected relative fluorescence (CRF). I f the known samples contained 0.4 ,~g. per ml.: 0-4 x CRF of unknown sample concentration of chloroqulne in unknown Mean CRF of known samples samples in ~g. per ml. An alternative method of calculation has been worked out. A range of concentrations of chloroquine base in 0.1 N HC1, from 0.1 to 1.0 ~g. per ml. was prepared. 2 mi. of each were made alkaline with 1 ml. of normal NH3OH, including a blank of 0.1 N HCI with no added chloroquine. These were read in the spectrophotofluorometer and the CRF of the samples plotted against concentration, giving a straight line passing through the origin. The CRF of unknown samples can be read on this graph, giving concentration in t~g. per ml. directly. This figure must be multiplied by 1.33 to give the true concentration, as 15 ml. of the original 20 ml. of heptane was taken in stage e above. It is considered that the first is the better method, as losses and errors incurred in the extraction process should cancel, but this is a useful check. A result by the first method that differs widely from that obtained from the graph would suggest a fault in the extraction of the known samples. R e c o m m e n d a t i o n s for collection o f s e r u m
a.
b.
c.
Two 5 ml. serum specimens should be taken before treatment begins, to give a blank value to be used for all estimations on that patient. A single blank specimen can be used but it is more accurate to use the mean of 2. All specimens should be kept in a refrigerator until examined to avoid high errors due to bacterial growth. On one occasion when the laboratory refrigerator broke down the specimens stored in it gave an impossibly high fluorescence on the chloroquine wave length, and extraction of a broth culture from these sera gave a similar result. It is not considered necessary to withhold salicylates. These do give an interfering fluorescence but do not come through the extraction process. REFERENCE
BRODIE,B. B. et al. (1947). J. Biol. Chem., 168, 319.
THE ESTIMATION OF CHLOROQUINE IN BLOOD SERUM BY
E. M. ENSOR* Formerly Officer-in-Charge, Research Wing, Far East Land Forces
In late 1962, cases of malaria in British and Commonwealth troops, who had been infected during jungle operations in southern Thailand and whose infections did not respond to the usual therapeutic course of chloroquine, began to reach British military hospitals in the Far East. Faced with this situation, it became necessary to estimate the blood chloroquine levels attained with the standard dosage, to determine if resistance to the drug was in fact present. Samples of serum from cases and controls were sent to the Central Pathological Laboratory, Singapore, and attempts were made to estimate the chloroquine in these according to the method described by BRODm et al. (1947). It was soon found that this method was giving very low values of chloroquine compared with what were known to be present, and the Research Wing, Far East Land Forces, was given the task of working out a reliable technique. A modification of the original method was described by Dr. Richard L. Jacobs of the Department of Health, Education and Welfare, Public Health Service, Bethesda, 14, Md., U.S.A., in a personal communication. In this method the chloroquine in serum is extracted into heptane with 5 N NaOH. The heptane is washed with 0.1 N NaOH and the chloroquine re-extracted into water with 0.1 N HC1. This is then made alkaline with ammonia, and the fluorescence due to the chloroquine is read in an Aminco-Bowrnan spectrophotofluorometer. The fluorescence from an unknown sample is compared with that given by a standard made by adding a known quantity of chloroquine to normal sel'unl.
A few modifications were made (mainly in increasing the time of the initial extraction to 45 minutes from the 30 originally recommended), and the technique finally evolved is as follows: a. A mechanical shaker is used which will take 9 fiat 4-oz. medicine bottles. These are loaded: (1) 2 with 5 ml. of normal serum (serum blanks); (2) 2 with 5 ml. samples of normal serum to which 0.4 ~g. of chloroqnine base per ml. has been added (known samples); (3) Each of the remaining 5 bottles with 3 to 5 ml. (3 is the minimum) of a serum specimen to be estimated (unknown samples). *The author's thanks are due to Professor Robert C. Y. Lin for his kind permission to use the spectrophotofluorometer and other laboratory facilities in the Department of Pharmacology, University of Singapore.
76
THE ESTIMATION OF CHLOROQUINE IN BLOOD SERUM
B ~ o ~
•~
•
~
0
o
bl~ • ~
~I
II
~ / o ~.~ ~o~ N~
0
0
0
(D
O
O
d .__qo
•~
~ ~0 ~
~t
o2 ¢..; ::~.,-~
,.o A
E. M .
ENSOR
77
..Q
e~
~bE
o.~
~OO
•
a
-
o.~
r'~
oa#
00
,,~
.
•
d °
0",
O
,,,-I
b.
c. d.
To each bottle is added 20 ml. of heptane, containing 1-5 ml. of isoamyl alcohol per 100 ml., and 1 ml. of 5 N NaOH. The bottles are then shaken for 45 minutes and the phases allowed to separate. The aqueous phase is discarded and 5 ml. of 0.1 N NaOH is added. The bottles are shaken for 10 minutes and the phases allowed to separate as before. As for c. above.
78 e.
£
THE ESTIMATION OF CHLOROQUINE I N BLOOD SERUM
15 ml. of the heptane phase are removed from each bottle and the remainder, with the aqueous phase, is discarded. The heptane is returned to the bottle and an amount of 0.1 N HC1 equal to the original volume of serum is added. The bottles are then shaken for 30 minutes. The heptane phase is removed by aspiration and discarded. 2 ml. of the aqueous phase is placed in the cuvette of the Aminco-Bowman spectrophotofluorometer and 1 ml. of normal NHsOH added and mixed by inversion. The samples are read, by setting the activating wavelength at 330 millimicrons and the fluorescence wave length at 390. The sensitivity control is kept at 40 throughout. Calculations
a.
b.
Percentage transmission x photomultiplier factor = relative fluorescence (RF). RF of samples, known or unknown, minus mean RF of serum blanks -=--corrected relative fluorescence (CRF). I f the known samples contained 0.4 ,~g. per ml.: 0-4 x CRF of unknown sample concentration of chloroqulne in unknown Mean CRF of known samples samples in ~g. per ml. An alternative method of calculation has been worked out. A range of concentrations of chloroquine base in 0.1 N HC1, from 0.1 to 1.0 ~g. per ml. was prepared. 2 mi. of each were made alkaline with 1 ml. of normal NH3OH, including a blank of 0.1 N HCI with no added chloroquine. These were read in the spectrophotofluorometer and the CRF of the samples plotted against concentration, giving a straight line passing through the origin. The CRF of unknown samples can be read on this graph, giving concentration in t~g. per ml. directly. This figure must be multiplied by 1.33 to give the true concentration, as 15 ml. of the original 20 ml. of heptane was taken in stage e above. It is considered that the first is the better method, as losses and errors incurred in the extraction process should cancel, but this is a useful check. A result by the first method that differs widely from that obtained from the graph would suggest a fault in the extraction of the known samples. R e c o m m e n d a t i o n s for collection o f s e r u m
a.
b.
c.
Two 5 ml. serum specimens should be taken before treatment begins, to give a blank value to be used for all estimations on that patient. A single blank specimen can be used but it is more accurate to use the mean of 2. All specimens should be kept in a refrigerator until examined to avoid high errors due to bacterial growth. On one occasion when the laboratory refrigerator broke down the specimens stored in it gave an impossibly high fluorescence on the chloroquine wave length, and extraction of a broth culture from these sera gave a similar result. It is not considered necessary to withhold salicylates. These do give an interfering fluorescence but do not come through the extraction process. REFERENCE
BRODIE,B. B. et al. (1947). J. Biol. Chem., 168, 319.