ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 9, No. 6 Copyright © 1979, Institute for Clinical Science, Inc.

Measurement of Salicylsalicylic Acid and Salicylic Acid in Plasma by High Pressure Liquid Chromatography

M ICHAEL M. LUBRAN, M.D., P h .D., STEPHEN N. STEEN, M.D., Sc.D.and ROBERT L. SMITH, M.A

Harbor-UCLA Medical Center Departments of Pathology and Anesthesiology

Torrance, CA 9050i

ABSTRACT

Salicylsalicylic acid (SS) and salicylic acid (SA) both appear in plasm a after the oral ingestion of the former. They can be estim ated in the presence of each other after extraction into dichlorom ethane using high pressure liquid chromatography. SS is unstable in plasma, being converted to SA in vitro. Conversion can be prevented by using ethylene diam inetetraacetic (EDTA) as an anticoagulant and carrying out the extraction in the cold. An aliquot of the extract is dried and dissolved in methanol. Weak solutions of SS in m ethanol are unstable at room tem perature. M ethanolysis can be prevented by the addition of dilute hydrochloric acid. By adding phenylbutazone to the plasma as an internal standard, variations in extraction and sample applica­tion are nullified. Quality control is achieved by concurrent extraction of two different plasma standards containing SS and SA in known amounts. About 40 ng of SS and SA can be detected; reproducibility at 200 ng and above is be tter than 6.5 percent.

IntroductionSalicylsalicylic acid (2-hydroxybenzoic

acid 2-carboxyphenyl ester) has been known since 19093 and is used as an analgesic. It is a white crystalline powder, almost insoluble in water. It is believed to act pharmacologically by conversion to salicylic acid. W here and how this conver­sion takes place is uncertain, bu t it may occur in the small intestine. Blood con­centrations of salicylsalicylic acid (SS) have been m easured indirectly, after its conversion to salicylic acid (SA) by acid h y d ro ly s is2 a t abou t 115°. P u b lish e d

pharmacokinetic studies of the drug have been based on total salicylate m easure­ments and not on SS concentrations.1,4,5 As a prelim inary to the investigation of the pharmacological action of SS, a specific m ethod has been developed, using high pressure liquid chromatography (HPLC), for the simultaneous determ ination of SS and SA in the presence of their m etabo­lites. The m ethod has been used to study the absorption of SS in normal subjects.

During the developm ent of the m ethod, several unexpected problem s occurred, the solution of which required a close

5010091-7370/1100-0501 $01.80© Institute for Clinical Science, Inc.

502 LUBRAN, STEEN AND SMITH

reexamination of extraction procedures as applied to HPLC. These problems and their solutions are described in detail in this paper as an aid to other investigators developing extraction methods involving HPLC. The method for the determination of plasma SS and SA concentrations de­scribed here includes some unique fea­tures, which provide quality control of the extraction and chromatography phases of the technique and greatly reduce the number of accurate measurements which must be made. The method in its final form will be described first; the problems and their solutions will then be discussed.

Principle

Salicylsalicylic acid (SS), salicylic acid (SA) and other substances are extracted with dichlorom ethane (m ethylene chloride) from acidified plasma to which phenylbutazone (PB) has been added as an internal standard. An aliquot of the extract is evaporated to dryness; the resi­due dissolved in acidified methanol and the SS, SA and PB concentrations of the solution determined by reverse phase HPLC. Appropriate standards are run.

Materials

Ap p a r a t u s

High pressure liquid chromatograph. In the work reported here, the HPLC ap­paratus* was fitted with a 6000A pump> U6K iryector and 440 detector. The col­umn was 30 cm long and 3.9 mm in inter­nal diameter. The supporting phase was MicroPorosil, of 10 ju.1 particle size. The stationary phase was Micro-Bondapak C 18, an organosilane bonded to the Micro­Porosil as a monomolecular layer. The mobile phase was methanol-acetic acid.

Rotator. Multipurpose Rotator Model 151 was used.f This instrument has vari­

* Waters Associates, Milford, MA 01757.t Scientific Instrum ents Inc., Lake Worth,

FL 33460.

able speeds and holds 16 tubes on a pla­ten, which can be fixed at any angle.

Waterbath. This was regulated at 50°.Extraction tubes. These were non­

disposable, 16 x 100 (or 125) mm, glass, screw-capped, with plastic liner resistant to organic solvents. Capped tubes must be leak proof.

Evaporation tubes. These were glass* disposable culture tubes 13 x 75 mm.

Pipets. Volumes up to 0.5 ml were m easured using SMI m icrop ip ets.$ Dichloromethane was delivered from a Repipet, 10 ml volume.?

R e a g e n t s

Dichloromethane. Certified ACS, B.P. 39.5°-40.4°. Store in dark bottle in cold room or refrigerator.

Methanol. Spectrophotometric grade. Store in dark bottle in the cold.

Dilute hydrochloric acid, approx. 0.27 mol per I. Twenty-five ml of concentrated hydrochloric acid (about 36 percent w/w) are diluted to one liter with distilled water.

Salicylsalicylic acid (2-hydroxybenzoic acid 2~carboxyphenyl ester).11

Salicylic acid. Certified ACS reagent.Phenylbutazone.1Glacial acetic acid. Certified ACS

reagent.Acid methanol. Two ml of dilute hydro­

chloric acid are diluted to 100 ml with methanol and stored in the freezer. The reagent should be prepared weekly.

St o c k So l u t io n s

P henylbutazone (20 m mol per I). Exactly 308.4 mg of PB are dissolved in methanol to a volume of 50 ml, transferred

| Scientific Manufacturing Industries, Emery­ville, CA 94608.

§ Labindustries, Berkeley, CA 94710.11 Obtained from Riker Laboratories, Inc., North-

ridge, CA 91324.H Sigma Chemical Co., St. Louis, MO 63178.

SALICYLSALICYHC ACID AND SALICYLIC ACID IN PLASMA 503

to several small glass screw-capped tubes and stored at freezer temperature (about =20®),

Salicylsalicylic acid (60 mmol per I). Precisely 774,7 mg of SS are dissolved in methanol to a volume of SO ml and treated as described previously,

Salicylic acid (100 mmol per I). Exactly 040,8 mg of SA are dissolved in methanol to a volum e of 50 ml and treated as described previously.

The stock solutions have proved to be stable in the freezer for at least 12 months. Nevertheless, they should be tested for homogeneity before use by suitable dilu­tion With acid methanol and examination by HPLC. The concentration of PB need not be known exactly, as it does not enter into the final calculation. The concen­trations of SS and SA must be known as accurately as possible, but can differ by as much as 5 percent from the values given previously, thus making it easier to weigh out the solids. The actual concentrations of the stock solutions used in the experi­ments were: PB 6,48 f i g per ml (21 mmol per 1); SS 15.30 f i g per ml (50.2 mmol per 1); and SA 13.45 f i g per ml (105.0 mmol per 1),

S t a n d a r d S o l u t i o n s

Plasm a sta n d a r d s—H igh (H ) and Low (L), Plasma is collected and prepared as w ill be described (collection of blood), It must be obtained fresh from volunteers who have fasted overnight and have not taken SS or SA for at least three days, About 55 ml of plasma are required. Small volumes of stock SS and SA are transferred into small glass tubes, which are covered and allowed to reach the temperature of the cold room (4e to 6e). The next steps are carried out in the cold room. About 15 ml of plasma are placed in each of two 25 ml volumeteie flasks (labelled H and L), To L are added 50 /u.1 of stock SA and 50 /¿I of stock SS; to H are added 150 f i \ of stocks A and 150 /u.1 of stock SS, The flasks are agi­

tated to dissolve the precipitate that forms and the volumes made to 25 ml with plasma. The contents of the flask are w ell mixed by repeated inversions and divided into aliquots of about 2.5 ml, which are kept in the freezer in small capped tubes. The concentrations of SS and SA will be 1/500 (for L) and 3/500 (for H) of their concentrations in the stock solutions.

Methanol standard for HPLC. Forty /ul of stock SA, 40 1 of stock SS and 300 fx 1 of stock PB are dissolved in acid methanol to a final volume of 10 ml. Nominal concen­trations are: PB 0.6 ju,mol per ml; SA 0.4 /u,mol per ml; and SS 0.24 f i mol per ml. Concentrations actually used (in f i g per ml) were : PB 194.4; SA 53.8; and SS 61.4.

O t h e r S o l u t i o n s

Working phenylbutazone (6 mmol per I). Fifteen ml of stock PB are diluted to 50 ml with methanol. The solution is stable for at least one month in the freezer.

Methanol-acetic acid (Mobile phase for HPLC). Distilled water is added to 600 ml of methanol to give a final volume of one liter. This solution is used to dilute 10 ml of glacial acetic acid to one liter. The solu­tion is prepared fresh and degassed by filtration through a 0.45n Millipore filter.

C o l l e c t i o n o f B l o o d

About 10 ml of blood are collected di­rectly into tubes containing EDTA anti­coagulant and well mixed. It is convenient to use Vacutainer* tubes containing the tripotassium salt of EDTA (and potassium sórbate) in solution, The tube is immedi­ately placed in the cold room, or in ice- water, and the plasma separated by use of a refrigerated centrifuge at 4s to 6s. Aliquots of the plasma are transferred to three small tubes which are capped and stored in the freezer.

* Bgeton & Dickinson, Rutherford, NJ,

504 LUBRAN, STEEN AND SMITH

Method

E x t r a c t i o n P r o c e d u r e

Test and standard (H and L) plasmas m ust be treated identically at all stages of the extraction. Duplicate test samples and a t least one H and one L standard are analyzed. A 16 place rotator allows seven test samples, one H and one L standard to be processed in a batch. Further test sam­ples are analyzed subsequently using the same rotator and add itiona l H and L standards. Once the optim al extraction time and the settings of the rotator have been determ ined, conditions m ust not be changed. The frozen plasma is allowed to thaw in the cold room, preferably while on the rotator to ensure thorough mixing.. Ex­traction tubes are labelled and into each is pipetted 0.5 ml of test or standard plasma, followed by 100 /xl of working PB solu­tion. The PB solution m ust be delivered directly into the plasma.

After thorough mixing of the plasma and PB to dissolve the precipitate which forms (mechanical agitation for 30 seconds), 300 jul of dilute hydrochloric acid are added to each tube and the contents mixing by tap­ping. (Acidification to about pH 1.5 frees the drugs from protein binding). After about three m inutes, 5 ml of dichloro- m ethane are added to each tube, which is tightly capped and placed on the rotator. The conditions and time for optimum ex­traction m ust be determ ined experim en­tally. In the work described here, the pla­ten was inclined at 30° from the vertical, the speed setting was 4 (about 20 r.p.m.) and the extraction time 45 m inutes.

Follow ing extraction, the phases are separated by centrifugation (in the cold room). Because SS, SA and PB are stable in dichloromethane at room tem perature, the rem aining steps are carried out at room te m p e ra tu re . T he u p p e r phase (plasma) is rem oved by aspiration (a small loss of the organic phase does not matter) and 1 ml o f th e su b n a ta n t phase (dichloromethane) transferred to an evap­

oration tube. Volumes o f organic phase are best m easured using disposable glass one ml pipets. Small errors in m easuring these volumes do not affect the result. The solvent is completely evaporated at 50° using a water bath (about 20 minutes). If HPLC analysis cannot be carried out at once, the tubes are sealed and stored in the freezer until required.

HPLC A n a l y s i s

The dried extract is dissolved in 200 /u.1 of acid methanol and the solution kept in the cold room until a few m inutes before injection into the HPLC apparatus. The operating conditions for the work de­scribed here were: wave length 280 nm, flow rate of mobile phase 2 ml per min, sensitivity 0.02 or 0.05, chart speed 1 cm per min and injection volume 20 ¿il or 10 /xl. U nder these conditions, SA, SS and PB peaks appeared in that order and were clearly separated. Retention times were approximately 2.6, 4.8, and 7.0 min, re­spectively, bu t varied slightly from run to run. In a typical experim ent, after equi­libration of the column with the mobile phase, two m ethanol standards were run, follow ed by the low plasm a standard, the te s t sam ples, th e h igh p lasm a standard and another m ethanol standard. At times, methanol standards would be interspersed w ith the test samples.

C a l c u l a t i o n

Peak heights above the baseline are m easured for SA, SS and PB in the test samples and plasma standards. They are also m easured for the m ethanol standards, but these values do not en ter into the cal­culation. Let the subscripts “cont” and “ p t” denote, respectively , the plasm a standard and test samples; the subscripts L and H denote low and high plasma standards. Let the peak heights of the drugs be SA, SS and PB and the concentra­tion of the drugs in the plasm a standards (in ju,g per ml) be cL amd cH for salicylic

SALICYLSALICYLIC ACID AND SALICYLIC ACID IN PLASMA 505

acid and dL and dH for salicylsalicylic acid. T he calcu lation , w hich also provides quality control, is carried out in four steps.

1. Calculate for each plasm a standard th e ratios (P B /S A )Lcont, (P B /S A )Hcont> (PB/SS)Lc0nt and (PB/SS)Hcont.

2. Calculate the ratios of ratios (PB/ SA)Lcont / (PB / SA)Hcont and (PB / SS)Lcont / (PB / SS)Hcont* These ratios should equal 3, w ith in a reasonable margin of error, be­cause the high standard is three tim es as concentrated as the low standard for each drug. A value betw een 2.5 and 3.5 is ac­ceptable, because the variances of each com ponent of the ratio of ratios are addi­tive.

3. Calculate the w eighted mean con­centration factor for the standards as fol­lows:c = 0.5 [cL (PB /S A )^^ + cH (PB/SA)hcoiJ d = 0.5 [dL (PB/SS)Lcont + dH (PB/SS)HCo„t]

4. The concentrations of the drugs in the test plasmas in f i g per ml are:S a licy lic acid c o n c en tra tio n (S) = c x (SA/PB)pt;Salicylsalicylic acid concentration (T) = d x (SS/PB)pt

If only one plasma standard is used, the calculation is

S = c* X (SA/PB)pt x (PB/SA)*;T = d* x (SS/PB)pt x (PS/SS)*,

where the * refers to either the low (L) or high (H) plasm a standard. Use of one standard elim inates the quality control feature of the method.

V a l i d a t i o n o f t h e M e t h o d

The somewhat elaborate procedure de­scribed previously was developed after considerable experim entation to reduce the many errors that can occur in an HPLC procedure that requires prelim inary ex­traction of a drug. For convenience, the problem s that arose and their solutions w ill be described in the o rder of the

analytical steps rather than in the order in which they were encountered.

S a m p l e S t a b i l i t y

The first blood samples analyzed were an tico ag u la ted w ith h e p a rin and ex­tracted at room tem perature. Sequential m easurem ents of a plasma sample spiked with SS (about 100 f i g per ml) showed a rap id conversion o f SS to SA at room tem perature, detectable after 15 m inutes and almost com plete in four hours. It was assum ed that enzymatic conversion o f SS- and SA occurred owing to non-specific es­terases, and an inhibitor was sought. Stor­ing heparinized plasm a in the cold room reduced the conversion but did not pre­vent it. Freezing heparinized plasma and allowing it to thaw at cold room tem pera­ture also led to considerable loss of SS.

As it was desired to inhibit loss of SS from th e m o m en t b lo o d was d raw n , ED TA was used as an an ticoagulant. EDTA was found to reduce SS loss mark­edly at room tem perature and to inhibit conversion of SS com pletely at cold room tem pera tu res. T haw ing frozen ED TA plasma in the cold room did not lead to loss of SS. In the work described here, blood was draw n into 10 ml capacity Vac- u ta in e r tu b es* co n ta in in g 15 mg of K3EDTA and 0.02 mg of potassium sór­bate in 100 ¿u.1 of solution. The small dilut­ing effect of the anticoagulant solution was ignored in the calculation: it produces an error of about 2 percent. Plasma was separated in the cold room and kept frozen until analysis. It was allowed to thaw in the cold room.

U s e o f a n I n t e r n a l S t a n d a r d

Internal standards are customarily used in HPLC to allow for errors in m easuring the volume of solution injected onto the column. They are also used in extraction procedures to monitor the efficiency of

* Beckton and Dickinson, Rutherford, NJ.

506 LUBRAN, STEEN AND SMITH

extraction. An internal standard added to the plasm a before extraction would serve both these purposes. The ideal substance should be readily available in pure form, easily added in exact amount to pi asma, be com pletely extractable or extractable in fixed am ount and, in the HPLC proce­dure, give a distinct peak near but not involving the drugs being analyzed or their metabolites.

The substance chosen as an internal standard, phenylbutazone, has all but one of these properties; it is not extracted com ple te ly from so lu tion b u t can be extracted in proportion to SA and SS if conditions are carefully standardized. To achieve this, it was found that the plasma standards and the test samples had to be processed as a batch, extraction being car­ried out on the same rotator. Optimum extraction of SA, SS and PB, i.e., constancy of SA/PB, SS/PB ratios, occurred in 45 min, which was therefore chosen as the extraction tim e. In this period, about 50 percent of PB was extracted. The effi­ciency of extraction varied from run to run, in spite of keeping conditions unchanged. Phenylbutazone was added in methanolic so lu tion . An aqueous so lu tion of the sodium salt would have been preferred, bu t this substance is not readily available. PB was found to be stable during the ex­traction and HPLC phases of the proce­dure.

C h o i c e o f W a v e l e n g t h

S alicy lic acid has an ab so rp tio n maximum at 300 nm, salicylsalicylic acid at 308 nm and phenylbutazone at about 230 nm (all in methanolic solution). As the instrum ent used employed an in te r­ference filter, one centered at 280 nm was chosen. At this wave length, an absor­bance of 1 is given by SA, SS and PB at concentrations of 91,138 and 18/xgperm l, respectively. The amount of PB added to the plasma samples was chosen to give a peak height of about 50 mm, under the analytical conditions of the test. PB con­

centrations and peak height w ere linear up to 200 fig per ml, S A up to 100 fig per ml and SS up to 100 fig per ml w hen 20 /u,l of the solution were injected and the sensi­tivity set at 0.05. Smaller volumes were injected for higher concentrations and for low concentrations a sensitivity of 0.02 was used.

C h o i c e o f E x t r a c t i n g A g e n t

A lipid solvent immiscible with water is required. Initially, hexane was used, bu t its relatively high boiling point (69°) proved inconvenient. D ichlorom ethane, which boils at 40° and is rapidly rem oved w hen tubes containing it are placed in a w ater bath at 50°, was chosen as the sol­vent. Because it is heavier than water, it forms the subnatant layer when the phases are separated by centrifugation in the cold. The upper layer is easily rem oved by suction. No error is introduced if some of the organic phase is lost during this pro­cedure, as only an aliquot is required. This is rem oved using a disposable glass one ml pipet. Because of the use of an internal standard, accuracy is not required at this stage.

C h o i c e o f F i n a l S o l v e n t

The residue from the dichlorom ethane ex trac t was o rig in a lly d isso lv e d in methanol and injected onto the column of the HPLC apparatus. It was found that the SS in the extract decomposed, the degree increasing as the sample was kept at room tem perature. SS was found to be stable during the evaporation phase. The de­composition of SS gave rise mainly to one or two substances with similar retention times, causing the appearance of shoul­ders on the SS peak. These were thought to be due to methyl derivatives caused by the action of methanol on the SS in very dilute solution at room tem perature. It was not possible to test this hypothesis owing to the nonavailability of these com­pounds. However, the addition of a small

SALICYLSALICYLIC ACID AND SALICYLIC ACID IN PLASMA 507

am o u n t o f h y d ro ch lo ric ac id to th e methanol used as solvent com pletely pre­vented this decomposition. Salicylic acid and phenylbutazone were not altered in methanolic or acid m ethanolic solution. Acid methanol standards used with the H P L C ap p a ra tu s w ere s to red in the fre e z e r an d a llo w ed to reach room tem p era tu re ju s t before use . U nused standard solutions were discarded.

S p e c i f i c i t y a n d Se n s i t i v i t y

SS and SA give peaks c lea rly d is ­tinguishable from those due to their m e­tabo lite s and o ther substances in the b lood . T he d eco m p o sitio n p roducts cau sed by m ethano l, w h ich p roduce shoulders on the SS peaks, are not found in extracts of blood obtained from subjects ingesting salicylsalicylic acid. However, if the stock solution of SS used for making the plasm a standards is not kept suffi­ciently cold, a shoulder may appear on the SS peak obtained from them. Extracts of blood from subjects not taking SS, SA or other drugs showed some peaks close to the point of injection, bu t they did not interfere with the SS, SA and PB peaks. T he m ethod has, th e re fo re , the h igh specificity associated with HPLC proce­dures. About 0.1 f i g of SA and 0.15 f i g of SS can be easily detected on the column at a sensitivity setting of 0.02.

R e p r o d u c i b i l i t y

T his was asse ssed from d u p lic a te m easurem ents over the analytical range of about 0.15 f i g per ml to about 15/j,g per ml in the original plasma. For each drug, the ratio of its peak height to PB (i.e., SA/PB and SS/PB) was determ ined for each dup­licate. The percent deviation of the values of the ratios from their mean value was then calculated. The results were: SA, 32 pairs, mean percent deviation 4.53, range 0.84 to 14.65; SS, 24 pairs, mean percent devia­tion 4.99, range 0.19 to 13.6. The devia­tions were asymmetrically distributed. All

the deviations greater than 6.5 percent oc­curred with samples of very low concen­trations, associated w ith peak heights less than 8 mm. W hen these resu lts w ere excluded, the percent deviation for SA (24 pairs) was 2.75 and for SS (17 pairs) was 2.88.

S o u r c e s o f E r r o r

Although only two volumes m ust be m easured with accuracy (the sample or s ta n d a rd an d th e w ork ing p h e n y l­butazone solution), the other volumes m ust be approximately correct to achieve m easurable peaks during HPLC and not to overload the colum n. P ositive d is­placement micropipets (e.g., SMI) are con­venient for batch work, are accurate and produce negligible carryover. However, they must be used w ith care. The glass capillaries should be changed frequently and seated firmly. Calibration should be checked (using the wire provided) each tim e a new capillary tube is used. The m icropipet should be tested for leaks at the start of each batch, and the plunger tip changed if necessary.

The addition of the phenylbutazone SO' lution could provide a major source of er­ror. All of the solution must be delivered into the sample and none left on the wall of the tube, and PB m ust be uniformly d istributed in the plasm a by thorough mixing. Small variations in the volume of d ilu te hyd roch lo ric acid add ed w ere found not to affect the amount of drugs ex tracted , p ro v id ed the pH was less than 2.

T he ex traction tec h n iq u e p rov ides another possible major source of error. To satisfy the basic assum ption that PB, SS and SA are extracted in fixed proportions of their concentrations in the plasma, it is essential that the standards and tests be extracted at the same tim e on the same rotator. It was found that discordant val­ues were obtained w hen standards were extracted on separate rotators. A rotator was used for extraction because of its con-

508 LUBRAN, STEEN A ND SMITH

Venience and the fact that, by its use, émulsions did not occur. Uniformity of ex­traction could not be achieved by mechan­ical agitation (Vortex); emulsions were occasionally produced by this procedure.

The sources of error in HPLC are well known and will not be detailed here. Be­cause the efficiency of the column de­teriorates during a long run and peak heights diminish, it is advisable to wash the column periodically. Errors owing to small differences in the volume injected do not affect the final answer.

Q u a l i t y C o n t r o l

The basic assumption of the extraction procedure is confirmed for each batch of ex trac tio n s by c a lc u la tin g th e ratios PB/SA, PB/SS for the low and high plasma standards. For each drug, the ratio of low to high standard should be 3. Because four m easurem ents of peak heights are in ­volved in this ratio of ratios, a range of 16 percent around 3 (2.5 to 3.5) was consid­ered satisfactory. In experiments on the abso rp tion o f sa licy lsa licy lic acid by healthy subjects (reported elsewhere), the values of the ratios were : for salicylic acid, mean 2.81, c.v. 5.2 percent, n = 10; for salicylsalicylic acid, mean 2.99, c.v. 4.4 percent, n = 10.

The use of acid methanol standards in the HPLC stage provides a check on the performance of the column. So long as the ratios of SA and SS to PB for the standard remain constant, the column is working reproducibly. W hen the peak heights start to decrease significantly , the colum n should be washed and reused when the peak heights of the standard have re­sumed their original value.

C a l c u l a t i o n

This takes into account the extractibilities of the drugs and the internal standard. To follow the derivation of the formula used for calculation, the m ethod will be sum­m arized: 0.5 ml of plasm a or plasm a

standard are used, plus 100 fi\ of phenyl­butazone; extraction is into 5 ml of sol­vent, of which 1 ml is evaporated to dry­ness; the residue is taken up in 200 jul of solvent and 20 /¿I of the solution are run by HPLC; 20 fi\ of acid m ethanol standard are run at the same time.

Let the concentration of added phenyl­b u taz o n e be P; of p h e n y lb u ta z o n e , salicylic acid and salicylsalicylic acid in the acid m ethanol standard be p, s, t, re ­spectively; the concentration of salicylic acid and salicylsalicylic acid in the plasma standard be c and d, respectively; and in the patien t’s plasma be S and T, respec­tively (all concentrations in fig per ml). Let the subscripts st, cont and pt indicate acid m ethanol standard, plasm a standard and patient’s plasma, respectively and let the peak heights be denoted by (PB), (SA) and (SS). Let the volume of solution in­jec ted onto the colum n be v^ul and, finally, le t E deno te the fraction of the drug extracted . In general, the fraction ex­tracted from different samples will not be the same. The basic assumption underlying the use of an internal standard is that for any sample, the ratio E pb/E sa is constant over the analytical range and similarly for the ratio E pb/E ss. Calculating these ratios for the plasma standard and the patien t’s plasm a and equating them leads to a sim­p le fo rm ula for th e c o n c en tra tio n of salicylic acid (S) and salicylsalicylic acid (T) in the patient’s plasma.

The calculation will be carried out for salicylic acid. The formula for salicyl­salicylic acid is similar.

F irst, consider the plasm a standard (cont). The dichloromethane extract con­tains, in fig per ml, (EPB)cont x P/50 of phenylbutazone and (ESA)cont x c/10 of salicylic acid; vcont therefore contains vcont/200 tim es th ese am ounts o f the drugs, in fig. The same calculation ap­plies to patien t’s plasma; the amounts of the respective drugs in vpt will be (vpt/ 200) X (EpB)pt x P/50 and (vpt/200) x ( E s a )* x S/10 fig. For the acid methanol

SALICYLSALICYLIC ACID AND SALICYLIC ACID IN PLASMA 509

standard, the respective quantities of the drugs in vst/u,l will be vst x p/1000 and vst X s/1000.

Next, consider the peak heigh ts. A linear rela tionship over the analytical range b e tw e e n peak h e ig h t and the am oun t o f d ru g c au sin g it has been e s ta b lish ed experim en tally . T hen , as (PB)st is due to pvst/1000 /tig of phenyl­butazone, (PB)cont is due to (PB)cont/(PB)st X pv^/lOOO ¡xg; similarly, (SA)COnt is due to (SA)cont/(SA)st x svst/1000/xg. Equating these respective quantities to the amount of drug applied to the column, we have:

(vcont/200) x (EPB)con, x P /50 =(PB)cont/(PB)st x pvst/1000 and

(vcont/200) x (ESA)cont x c/10 =(SA)cont/(SA)st x svst/1000.

From these equations, (EPB/E SA)cont =(PB/SA)cont x (SA/PB)st x 5 pc/Ps.

Similarly, for the patient’s plasma (pt):(E PB/E SA)Pt =

(PB/SA)pt x (SA/PB)st x 5 pS/Ps.

The basic assumption states that these two values of the ratios of extraction frac­tions are equal. By equating them and after some sim ple algebra, we obtain the desired result:

S = c x (SA/PB)pt x (PB/SA)cont Similarly, T

<1 x (SS/PB)pt x (PB/SS)cont ¿ig/ml.

It will be noted that only the volumes of plasma and added phenylbutazone solu­tion enter into the calculation. The con­centrations of salicylic acid and salicyU salicylic acid in the plasma standard must be known exactly, but not their concen­trations in the methanol standard.

As two plasma standards of different drug concentrations are used, the values of c x (PB/SA)cont should be the same for each; similarly for d x (PB/SS)cont. In prac­tice, they differ. Therefore, the best esti­mate of the standard concentration of each drug is given by the w eighted mean value, calculated as described previously.

Discussion

The inconvenience of working in the cold room would be avoided if a suit­able esterase inhibitor could be found. F luo ride , a know n in h ib ito r of some nonspecific esterases, will be studied in future experiments. The slowest step osf the p rocedure is the H PLC stage, in which about six samples an hour could big processed, thus lim iting to 14 the num ber o f p a tie n t sp ec im en s th a t co u ld be examined in a day. However, this work load could be tripled by use of an automar tic sample injector. In some of our work, the WISP injector* was successfully used. Thorough cleaning of the extraction tubes is necessary, to prevent artifacts on the recording. A final rinse in m ethanol wag required to remove traces of organic com­pounds causing spurious peaks.

Application of the analytical procedure in the study of oral adm inistra tion of salicylsalicylic acid to healthy subjects has revealed the presence of both salicyl­salicylic acid and salicylic acid in the plasma. Each drug reaches a peak concert- tration, salicylic acid peaking later than salicylsalicylic acid. This finding is con­sistent w ith conversion of salicylsalicylic acid to salicylic acid after absorption, al­though the possibility of hydrolysis of the drug in the small in testine cannot be excluded. These possibilities are being investigated.

P ub lished reports o f salicylsalicylie acid and salicylic acid concentrations in blood following oral adm inistration of salicylsalicylic acid m ust be interpreted with caution, as the investigators have ap7 parently been unaware of the rapid loss of blood salicylsalicylic acid, once the blood has been drawn. No precautions to pre­vent this loss are given. The analytica} methods used employ either fluorometry or the color reaction with ferric salts. Both methods give erroneous results if, as in some blood specim ens, salicylic acid and

* Waters Assoc.

510 LUBRAN, STEEN AND SMITH

salicylsalicylic acid are present together. M ethods involv ing acid hydrolysis of salicylsalicylic acid at a high tem perature, followed by m easurem ent of total salicy­late may also give rise to errors, because of the concomitant hydrolysis of the princi­p le m etabolites of salicylic acid.

Acknowledgm e nt

Thanks are extended to Patricia Anderson and Sandra Smith for technical help and Dr. O. Re of Riker Laboratories, Inc., for the supply of salicyl­salicylic acid.

References1. F r e n c h , I. W . and M il d o n , C. A.: Salsalate

(S-S) and a cety lsa licy lic acid: overn ight maintenance of therapeutic serum salicylate levels. Clin. Therapeut. 1 :353-358, 1978.

2. KOZJECK, F.: University of Ljubljana, Yugo­slavia. Personal communication.

3. M e r c k I n d e x . 9th edition 1976. Entry no. 8096,4. N o r d q u i s t , P., H a r t h o n , J . G. L., and

KARLSSON, R.: Metabolic kinetics of salicyl­salicylic acid, aspirin and sodium salicylate in man. Nord. Med. 28:1074-1076, 1965.

5. RUBIN, H. S.: Serum salicylate levels in osteo­arthritis following oral administration of a prep­aration containing salicylsalicylic acid and acety lsa licy lic acid . Amer. J. Med. Sei. 248:31-36, 1964.