CLIN. CHEM. 20/2, 152-158 (1974)

152 CLINICAL CHEMISTRY, Vol. 20, No.2. 1974

Furosemide Concentrations in Serum and Urine, and Its Bindingby Serum Proteins as Measured Fluorometrically

A. W. Forrey, B. Kimpel, A. D. Blair, and R. E. Cutler1

We describe a modification of the fluorometric meth-od of H#{228}usslerand Hajd#{252}[Arzneim. Forsch. 14, 704and 709 (1964)] for assay of furosemide in eitherserum or urine. A 1-mi sample, acidified to pH 2, isextracted with 5 ml of diethyl ether; 4 ml of the etheris back-extracted into 1 ml of phosphate buffer (pH7.0, 0.1 mol/liter), and finally acidified with 1 ml ofdilute HCI (0.6 mcI/liter). A procedure for estimatingblanks in urine was derived to correct for dilutioncaused by diuresis. Internal standards are used, andthe “effective” extraction ratio is used to correct forthe effects of quenching and extraction differences.In equilibrium, 93% of the drug is bound to serumproteins; 65% is tightly bound. Erythrocytes containless than 5% of the drug. Quantum yield of fluores-cence at pH 1 is 0.0496 for furosemide and is0.0163 for 4-chloro-5-sulfamoylanthr#{224}nilic acid.Furosemide fluorescence diminishes with increasingpH, while that of 4-chloro-5-sulfamoylanthranilicacid (a degradation product) increases.

Additional Keyphrases: pharmaco kinetics #{149}drug bind-ing by serum proteins #{149}colorimetry compared

Hajd#{252}and H#{228}ussler(1) and H#{228}usslerand Hajd#{252}(2) reported a fluorometric assay for furosemide (anonmercurial diuretic of the sulfonamide group) for

use in measuring concentrations of the drug inserum. We find that this method is subject to severalsources of error not previously noted and have there-fore modified the technique. In earlier studies of theurinary excretion of furosemide, H#{228}usslerand Hajd#{252}(2) used the colorimetric determination of urinary

furosemide concentrations by employing the Brat-ton-Marshall reaction for the detection of aromatic

amines. We have also applied the fluorometric meth-od to urine samples because a direct comparison ofthe concentrations in serum and urine is required fordata on plasma and renal clearances of the drug; itseemed preferable to use the same fluorometric

Department of Medicine, University of Washington at Harbor-view Medical Center, Seattle, Wash. 98104.

1 Address reprint requests to: R. E. Cutler, M.D., 325-9th

Ave., Seattle, Wash. 98104.Received July 30, 1973; accepted Sept. 10, 1973.

method, not only for convenience, but also to mini-mize systematic errors. We have also studied thebinding of the drug to serum proteins, in order to in-terpret its pharmacokinetics.

Materials and Methods35S-labeled and chemically pure unlabeled furo-

semide were obtained by courtesy of Hoechst Phar-

maceuticals Inc., Somerville, N.J. 08876.

Spectra

Absorption spectra were recorded with a “Spec-tronic 505” spectrophotometer (Bausch & Lomb,Rochester, N. Y. 14603) in 0.1 mol/liter sodiumphosphate and acetate buffers at selected pH’s. Un-

corrected measurements of fluorescence excitationand emission spectra were made in a Model 430spectrofluorometer fitted with a scanning attach-ment (G. K. Turner Associates, Palo Alto, Calif.

94303). For this, samples were prepared in thesesame buffers,but 10-fold more dilute than the con-centrations used for absorption spectra.

Quantum yields of furosemide (4-chloro-N-furyl-methyl-5-sulfamylanthranilic acid) and CSA2 emis-sion were measured in the same instrument, with useof quinine bisulfate standards, by the procedure ofParker and Rees (3). The samples, in buffers of vari-ous pH, and the quinine standard, in H2S04 (0.1mol/liter) at the same absorbance (within 0.05) asthe drug at the exciting wavelength, were excited at345 nm and the emission spectra were recorded. Ef-fective quantum yields were calculated from the rel-ative areas of the intensity vs. frequency spectra.

Assay

The method of H#{228}usslerand Hajd#{252}(2) was modi-fied as follows: 1 ml of serum or urine was acidifiedwith about 50 sl of concentrated HC1 and extractedfor 2 mm with 5 ml of diethyl ether in a 16 X 150mm screw-capped culture tube by shaking rapidly on

2 Nonstandard abbreviation used: CSA, 4-chloro-5-sulfamoyl-

anthranilic acid.

B, =

CLINICAL CHEMISTRY, Vol. 20, No. 2, 1974 153

a wrist-action shaker, and then centrifuged. A 4-mlaliquot of the ether phase was removed and re-ex-tracted for the same time interval with 1 ml of 0.1mol/liter potassium phosphate buffer, pH 7.0, fol-

lowed by centrifugation, and the ether was then re-moved. One milliliter of 0.5 mol/liter HC1 was thenadded and the fluorescence read in a Turner 430spectrofluorometer set at 345 nm excitation and 417

nm emission, with 60-nm bandpass.A control serum was obtained immediately before

furosemide administration. Normal sera containing1, 5, and 10 mg of furosemide per liter, internal stan-

dards, and aqueous standards of 0.5, 1, 2, 3, 5, 7, 10,and 15 mg/liter were taken through the same proce-dure. The working standards were prepared from thesame lot as the drug administered. These were, inturn, standardized against a pure furosemide stan-

dard solution. This procedure provides a control of

the amount administered, because furosemide de-grades slightly to a relatively nonfluorescent product,CSA, during storage, injection, and analysis.

Urine controls were prepared from a timed basalurine sample obtained from the study subject imme-diately before furosemide was administered. Thispre-drug urine was diluted in proportion to the ratiobetween the pre-drug and post-drug urine flow rate.To demonstrate the validity of this correction proce-dure, we compared a blank curve of serial dilutionsof the pre-drug urine in one normal subject withtimed samples obtained during an ethacrynic acid-

induced diuresis.Ethacrynic acid was chosen be-cause it closely mimics the diuretic response seenwith furosemide, but has no measurable fluorescenceat concentrations as high as 100 mg/liter. The rela-tionship between the measured blank value and theurine flow rate is described by the equation:

B0F0.(V,-35)+(B,135)

yr E

where B = blank urine sample, i; B0 = blank of pre-drug urine sample; F0 = flow rate of pre-drug urine;V = volume of urine sample i; T1 = collection time

or urine sample i, in minutes; B1 = blank urinesample immediately preceding sample i; and E =

“effective” extraction ratio.This estimated blank3 value is a ratio of the drug-

induced urine flow rate to the pre-drug flow rate,

with an adjustment made for a urinary tract (renal

pelves, ureters) hold-up volume of about 35 ml.We controlled the “effective” extraction ratio by

adding internal standards to both the serum andurine samples, and compared the properly correctednet signal caused by the internal standard to that ofthe equivalent unextracted acidified aqueous stan-dard. The sample extraction ratio was calculatedand then used in conjunction with the sample values

3This is an estimated value based on a post-micturition volumeof residual urine in upper and lower tracts of 20-50 ml. Residualvolumes may be even larger during diuresis when urine flow ratesare high.

to predict the concentration from the least-squaresfitto the straight line of the unextracted aqueousstandards. These calculations and further computa-

tions, which express the final results in terms of thepercentage of injected dose present in each liter ofserum, were done on a Burroughs-5500 computerusing a program written in the ALGOL program-

ming language.

BindingWe used the method of Hummel and Dreyer (4) to

measure equilibrium binding by collecting samplesfrom a 0.9 X 35 cm column containing “SephadexG-50” (Pharmacia, Piscataway, N. J. 08854), whichwas eluted at 12 ml/h with a special “binding buff-

er” having the same ionic concentration as serum(NaHCO3, 16; NaCl, 138; NaH2PO4, 4; KC1, 4;CaCI2, 2.5; and MgCl2, 1.5 mmol/liter), and adjust-ed to pH 7.40 with HC1. The column was equilibrat-ed with buffer plus the desired concentration of furo-semide and any potential competitors such as salicy-lates. A 0.5-ml aliquot of serum was carefully layeredat the top of the resin bed, under the buffer, with a15-cm 16-gauge needle and a 2-ml syringe. Forty

2-ml fractions were collected and analyzed in one oftwo ways: (a) chemically, either by the direct acidifi-cation of 1-ml aliquots of the nonprotein containingfractions or by ether extraction of the protein-con.taming fractions and several adjacent nonprotein

containing fractions; or (b) radiochemically, when35S-labeled furosemide was used, by pipetting 1 mlinto a 20-ml vial for liquid scintillation counting asnoted below. The net area under the peak, expressedin milligrams, and that contained in the trough werecalculated and the furosemide bound to a literof

serum protein was calculated from the average area.The percentage of drug bound per liter of serum is:

Binding(%) 100\ L’.-’eql

where A = mg of drug bound per liter of serum, andCeq = equilibrium concentration of unbound drug, inmilligrams per liter.

An identical unequilibrated separation on the col-umn was also conducted with a 0.5-ml serum sampleto which furosemide was added to give a total serumconcentration identical to that of the free drug in thebinding buffer of the equilibration column (10 mg/liter). The samples from this column were analyzedas above. Binding of furosemide by serum proteinswas also measured with “Centriflo” filter cotes (Am-icon, Lexington, Mass. 01273), which had beenshown to give a constant ratio of furosemide in thefiltrateto that in the nonfiltrable material upon re-peated trials, with use of furosemide dissolved inbinding buffer. Binding to erythrocytes was estimat-ed on aliquots of heparinized whole blood to which[35S]furosemide at a concentration of 10 and 15 mg/liter of whole blood had been added and equilibratedfor 15 mm. The packed erthrocytes were isolated by

S

Fa,

#{188}00

1,

furosem,de

9jin’ne

4chIoro- 5- sulfamoylanthronilic acid

Wavelength - nm

#{149}F.,owNd.o 4- ddo,o- 5. $uIf,nOyIOflth,OnhI,c id

A,thonihC ocd.06

05

04ba,

03

02

3 4 5 6 7 8 9

Butter pH

I0

Fig.2. pH dependence of furosemide and 4-chloro-5-sul-famoylanthranilic acid, with that of anthranilic acid shownfor comparisonThe samples are of equal absorbance and are diluted shortly beforereading to diminish acid catalyzed degradation of turosemide

Results Assay

154 CLINICAL CHEMISTRY, Vol. 20, No. 2, 1974

centrifugation and the plasma fractions saved foranalysis. The cells were then twice re-suspended intwo plasma volumes of isotonic saline and re-centri-fuged. The supernatant solutions were decanted and

saved, and the cells were finally resuspended in oneplasma volume of saline. Furosemide concentrationswere determined in all fractions, both by fluoromet-nc analysis and by liquid scintillation counting of

the serum, by the assay procedures described. Thechemical methods were controlled by adding internalstandards to the aliquots of serum and wash solu-tion.

Thin-Layer Chromatography

Thin-layer chromatograms were run on “Q-1” sili-

ca gel plates (Quantum Industries, Fairfield, N. J.07006), using the solvent system of Kindt andSchmidt (5) and that of Diamantstein and Ehrhart(6). The plates were examined under long wave-length (366 nm) ultraviolet light and sprayed withBratton-Marshall reagent as described by Kirschner(7).

Liquid Scintillation Counting

One-milliliter serum samples were mixed with 1ml of “Protosol” (New England Nuclear, Boston,

Mass. 02118), on a vortex-type mixer, afterwhich 15ml of “Aquasol” (New England Nuclear) was added

and again vortex mixed to give a clear homogenoussolution.A seriesof quench standards were preparedfrom 100 &l of aqueous [35S]furosemide standard, to

which 1 ml of water or 1 ml of serum and various

concentrations of erythrocyte hemolysate was addedas quencher. The samples were counted on a Model3315 liquid scintillation spectrometer (Packard In-

strument Co., Inc., Downers Grove, Ill. 60515) at19% gain and window settings of 50-1000 and 250-1000. The AES channel was set at 2.5% gain 300-1000. The counts, recorded on punched paper tape,were quench corrected off-line as described by Forrey(8).

Spectra

The excitationand emission spectra of furosemideexhibited maxima at 342 nm and 417 nm, respective-ly, in agreement with the values of HajthI andH#{228}ussler(1). The excitation and emission spectra ofCSA in 0.1 mol/literHC1 is shown in Figure 1. The

quantum yields of furosemide and CSA at pH 1.0were 0.0496 and 0.0163, respectively. The relation-ship between pH and fluorescence intensity for bothfurosemide and CSA is shown in Figure 2. Thesevalues demonstrated that the fluorescence emission

of furosemide is enhanced at acid pH’s compared tothat of the CSA, which is present as a contaminatingbreakdown product in all preparations of furosemide.Likewise, the relativesensitivityof the fluorescenceof these compounds to changes in solvent pH is ap-

parent, and is especially important when ion-ex-

Fig. 1. Emission spectra of 4-chloro-5-sulfamoylanthranil-ic acid and furosemide, excitedat 340 nm in 0.1 mol/liter HCI and recorded immediately after dilutionThe samples are of equal absorbance at the exciting wavelength. Thequinine spectrum is scaled by a factor of 0.1 with respect to the othertwo spectra and was recorded from a sample in H2S04 (50 mmol/liter)

change column chromatography is used to separatethese compounds before their concentration is mea-sured fluorometrically.

When we first used the assay reported by H#{228}usslerand Hajd#{252}(2), we had difficultyin re-dissolvingtheresidue after evaporation of the ether extract. Back-extraction of furosemide into neutral phosphate buff-er circumvented this difficulty in all cases, while si-multaneously diminishing the background fluores-

cence in urine samples by 40%.The first factor of importance in adopting this

modification was to assess how reproducible the ex-traction and back-extraction processes were. The

amount of drug remaining in the aqueous phase wasmeasured after ether extraction of acidified water,serum, and urine samples. The furosemide remainingin the ether phase of these same samples after back-extraction into neutral phophate buffer was alsomeasured. Two approaches were taken: (a) the

slopes and intercepts of the fittedstraight lines to

Regression ilnea

Sample type

Unextractedaqueous std.

No.

samples

63

intercept mg/liter

-0.102 ± 0.020

Slope,mg/liter/division Blank reading,b divisions

X 10’

5.89 ± 0.13 0

7.60 ± 0.99

0

10.00 ± 2.32

(range 0.02-22.04)

7.48 ± 1.23

Effectiveextraction

ratiocalculatedfrom slop.

ratios#{176}

0.93

0.72

0.71

Effectiveextraction ratio5

0.88 ± 0.01(range 0.69-1.10)

0.71 ± 0.02(range0.41-1.00)

0.56 ± 0.02(range 0.20-0.88)

#{149}diluted p.ediurCic uoine#{149}opine dor.nQ ethocrynic oc,d di,o,.wl

- calculated d,lotiOfl

‘.05

0.3IL-

a

0

Fig. 3. Dilution effect of ethacrynic acid-induced diuresison fluorescence of urine blanks carried through the entireanalytic proced ure

Flow Rate Ratio (Vs / Vo I

II

a)

\0,,E

10 50Meter divisions

150

Fig.4. Standard curves of extracted and unextracted fu-rosemide are shown as a regression of concentration onmeter readingExtracted (- -) slope, in mg/liter/div.: 0.0612 ± 0.00077: intercept Inmg/liter: 0.085 ± 0.260; standard error ot tlt: 0.135. Unextracted (--)

slope: 0.0559 ± 0.00047; intercept: 0.225 ± 0.114; standard error of fit:0.071

Table 1. Characteristics of Furosemide Assay

CLINICAL CHEMISTRY, Vol. 20, No. 2, 1974 155

Ether-extracted 63 -0.298 ± 0.022 5.47 ± 0.13aqueous std.

Ether-extracted 53 -0.120 ± 0.093serum andinternal std.

Ether-extracted 46 -0.105 ± 0.104 7.75 ± 0.35

urineandinternalstd.

Mean ± SD.Mean ± SEM.

Calculated from ratio of slope of unextracted aqueous standards to slope of extracted sample.

the fluorometric measurements of the extractedstandards in water, serum, and urine were comparedto those of the unextracted drug in acidified water;and (b) the measured radioactivity appearing ineach of the fractions before fluorometry was com-pared. The back-extraction process significantly di-minished the variable blank values for urine samplesbut, because furosemide is a diuretic, the fluorescentimpurities were variously diluted in each urine sam-ple. Because a true blank value is unmeasurable dur-ing furosemide diuresis, an estimated value is re-quired so that the sample can be corrected for suchdilution.4 We estimated the true blank values byassaying urine samples obtained at various flow ratesduring an ethacrynic acid diuresis (see “Methods”section). Dilutions calculated from the ratio of pre-drug to post-drug urine flow rates gave a good fit tothe observed data (Figure 3), whereas calculationsmade from changes in osmolality gave a poor fit.This result might have been predicted from theknown effect of ethracrynic acid and furosemide onrenal concentration and dilution mechanisms. Itshould be noted, however, that this model for theurinary measurement of furosemide assumes that thedrug has no effecton either the reabsorption or thesecretion of fluorescent impurities, but only on theurine flow rate, thus producing only a dilution ofthese substances. With the aforementioned correc-tions, our assay results for furosemide extracted fromwater, serum, or urine samples are respectively lin-ear (Table 1).

Over the calibration range 0-15 mg/liter, theblank corrected values for the extracted aqueousstandards do not fit the same regression line as theunextracted aqueous standards, as shown by the pa-rameters of the fit of these lines (Figure 4). The ratio

The blank value is the signal that would be produced by theinstrument if it were measuring a sample produced by a compa-rable urine flow rate in the absence of the diuretic furosemide.

No. samples

276

Effective extractionratio’

0.71 ± 0.020.80 ± 0.02

0.29 ± 0.03

156 CLINICAL CHEMISTRY, Vol. 20, No.2, 1974

of these slopes does agree reasonably well, however,with the “effective” extraction ratios for both aque-ous and serum samples. Moreover, the radiochemi-cally measured partition coefficient for serum sam-ples agrees closely with the extraction ratios mea-sured fluorometrically.

The differences noted above between the slopes oflinesfittingthe aqueous extracted standards and the

internal standards that are added to urine andserum is related to a value expressed as “effective”extraction ratio. This value is composed, in part, of apartition coefficient of the drug between the etherphase and the back-extraction buffer. In urine, thispartition may vary because each patient will excretedifferingconcentrations of the various solutes thatare present in urine. These substances would tend toincrease the solubility of the drug in the ether phase,particularly during the back-extraction, owing to theaffinity of the uncharged but somewhat polar solutesextracted into the ether for the furosemide and theaffinity of the aromatic compounds for the organicphase. This partitioning effect is demonstrated when1.0 mol/liter vs. 0.1 mol/liter of neutral phosphatebuffer is used to back-extract aqueous standards(Table 2). In this example, the partition coefficientas measured radiochemically between aqueous,serum, and urine standards is in the direction pre-dicted, but it is not as dramatic as the change in the“effective” extraction ratio measured fluorometrical-ly.While the “effective”extractionratiobetween ex-

tracted internal standards and unextracted aqueousstandards can vary between assays, the standard de-viation of the within-run error ratio remains constantat about 3%. It was also observed-by radiochemicalcounting and by thin-layer chromatography of theconcentrated aqueous phase of extracted aqueousstandards and urine samples-that little residual fu-rosemide or CSA could be detected in the acidifiedaqueous phase, either on thin-layer plates sprayedafter development with Bratton-Marshall reagent ormeasured directly as radioactivity. These findingsagree with those noted by Kindt and Schmidt (5).

Table 2. Extractable Radioactivity When[S]Furosemide is Added to Serum and Urine

and Back-Extracted into Different PhosphateBuffers

Sample type

0.1 mol/literneutral phosphate

buffer

serumurine

1.0 rnoi/liter

neutral phosphate

buffer

serum 12

#{176}Mean ± SD.

Likewise, only small amounts of drug could be de-tected chemically in the ether phase of extracted

aqueous or serum standards after the back-extrac-tion into neutral buffer, but urine samples behaved

differently.Nonetheless, although most of the drugin samples was back-extracted (as measured radio-chemically and judged by the absence of either flu-orescent spots or Bratton-Marshall reacting materi-als from the ether phase after thin-layer chromatog-raphy), the measured fluorescence intensity attribut-able to the drug was lower, particularly with urinesamples, than for the same concentration of furosem-ide in the neutral buffer measured directly after

acidification.We thought that possibly the acid catalyzed a

transformation of furosemide to CSA, producing alower fluorescence. Additionally, the quenching ef-fect of either dissolved organic solvent or peroxidecould be responsible. The possible quenching effectof peroxide was studied by warming the neutral buff-

er solutionsat 37 #{176}Cin the dark for 20 mm and thenadding mercaptoethanol, ascorbic acid, or stannouschloride as reducing agents to counteract the possi-ble quenching effects of peroxide. Such measures didnot change the results, however, suggesting that per-

oxides are not involved. Possibly other extracted in-terfering substances may be operating as internal fil-

ters and (or) chemical quenchers.

Because all of the major errors in furosemide esti-

mation in clinical samples, particularly in urine, are

reflected in the “effective” extraction ratio, attemptswere made to control this potential source of diffi-culty by adding internal standards to selected urinesamples. Typically, the samples used were early orpre-administration samples, with little or no furo-semide, respectively, in order to avoid errors of mea-

suring small differences in relatively large values

that result from the peak urine collections in oralstudies or early collections in intravenous studies.The calculated furosemide concentrations for thesesamples are related by the extraction ratio of the

urine sample to the unextracted aqueous standard.

The extraction ratiosfor these early samples may befurther used for subsequent dilute urine samples.The results (Table 1) demonstrate that “effective”extraction ratios for urine samples are substantiallylower than those of comparable saline standards, al-though the radiochemically measured partition coef-

ficients are similar. Also, urinary concentration aregenerally subject to considerably greater variation in

a given experiment series. Internal standards addedto serum samples also give somewhat lower “effec-tive” extraction ratios, but effect the serum concen-tration curve much less.

Interesting results relevant to “effective” extrac-tion was discovered in the urine of patients withrenal insufficiency who were receiving large quan-tities (2 mg/kg per day) of furosemide for therapy.When 240 mg of [35S]furosemide (specific activity, 80nCi/mg) was given intravenously to those patients,as much as 36% of the urinary radioactivity could

0 (2,606,782)

10 20 30Fraction number

Fig.5a.Chromatographyof fresh serum equilibrated with[355]furosemide at 10 mg/liter in binding buffer (condi-tions described in the text)Numbers adjacent to the peak and trough are total Counts bound per mil-liliter of serum: figure on the plateau is the concentration of the free hg-and, in cpm/ml

l8I’

0z_m3

PROTEIN

#{149}-.Furosemide #{149}SerumFurosemide

10 20 30 40Fraction number

Fig. 5b. Separation of free and rapidly equilibrating furo-semide from strongly bound drug, at 10 mg of total furo-semide per literThe area of the protein peak is 65% of the total count. The columns arerun under the identical conditions depicted in Figure 5a but without higandin the buffer. Adsorption phenomena probably explains the later elutiontime of the tree ligand compared to the figure above

experi.Type of experiment ments

Need for a convenient and relatively precise meth-od in which common laboratory instruments areused to determine furosemide in biological fluids has

No. led to the use of the fluorometric technique. ThisPercent bounda method is adequately sensitive and reasonably selec-

tive for use with clinical samples; its main drawback12 0.95 ± 2 is that there may be interferences from other fluo-

2 0.91 ± 1 rescent substances that may also be present in these

samples. With care,the use of solventextractionprocedures can lead to reasonably reliable measure-

2 0.68 ± 1 ments of un-transformed drug. CSA, a contaminat-ing or biotransformed product, causes insignificantfluorescent interference, owing to its low quantum

1 0.84 ± 1 yield under the conditions of this assay.This simple assay is unsuitable for measuring

metabolic products. The effects of pH, solvent parti-tioning, different quantum yields, superimposed ab-

2 0.040 ± 0.1 sorption peaks, fluorescing impurities, and quenchersof fluorescence dictate a preliminary separation be-

________________ fore measurement. Recently, a method in which

CLINICAL CHEMISTRY, Vol. 20, No.2, 1974 157

not be extracted by organic solvents from acidifiedsamples, even after repeated trials. These com-pounds would not be detected by the present assayand would explain, in part, the lower urinary excre-tion of un-transformed furosemide observed in these

patients as compared to normal subjects (9, 10).

Binding

The nature and extentof binding of furosemide toserum proteins was examined by the equilibratedcolumn technique of Hummel and Dreyer (4); weused both [35S]furosemide and fluorometric analysis

to measure the concentration. Although less precise,the chemical determinations substantially agreedwith the more precise radiochemical determinations.The extent of equilibrium binding, as determined bythis technique in two concentrations of ligand, isshown in Table 3. To obtain some gross measure-ment of the dynamics of binding in whole serum, fu-rosemide was also added to serum and the mixturewas separatedon the same column ofG-50 Sephadexused above, which was equilibrated with bindingbuffer only. As shown in Figure 5, the well-separatedunbound furosemide peak contained only 35% of thetotal drug while, at the time of elution (20 mm), theprotein fraction still had 65% of the added furosem-ide, indicating that the half-time for dissociation ofone class of slow binding sites probably exceeds 30

mm, while that of another class of fast sites is proba-bly less than 1 to 2 mm. The extentof equilibriumbinding, as measured by ultrafiltration with Amicon

Centriflo filter cones (Table 3), was 95%, in goodagreement with measurements by the technique ofHummel and Dreyer. Because studies with free furo-

semide in binding buffer indicated that some furo-semide was bound to the filtercones, the precision ofthis estimate is somewhat lower, owing to both ran-dom and systematic error.

The extent of furosemide binding to erythrocyteswas estimated by adding [35S]furosemide to wholeheparinized blood. Recovery of furosemide from the

Table 3. Binding Properties of [S]Furosemide

Serum“Centriflo”filter cones

“G-50 Sephadex”columnequilibrated with[Slfurosemide, 5 mg/liter

Binding buffer onlyusing serum with[S]furosemide, 10 mg/liter

Equilibrated with[t5Sjfurosemide, 5 mg/liter,and sodium salicylate,100mg/liter

Erythrocytes

u Mean ± SD.

serum and washes (Table 3) was 96% of the doseadded, indicatingthat the extentof erythrocytese-questration of furosemide, as determined by thismethod, is certainly considerably less than 10%.

Discussion

158 CLINICAL CHEMISTRY, Vol. 20, No.2, 1974

high-pressure liquid chromatography is used hasbeen developed in our laboratory to separate andmeasure furosemide in unextracted serum and urinein effluents of ion-exchange columns by both fluoro-metric and spectrophotometric detection techniques,with comparable sensitivity.

The present method for measuring furosemide inserum is straightforward, requires only common lab-oratory instrumentation, and can be adapted forclinical use in monitoring these concentrations in pa-tients. However, in patients with normal renal func-tion, serum concentrations of only 1-2 mg/liter pro-duce effective diuresis and the drug is rapidly elimi-nated (serum half-life, about 30 mm), with 85-95%of injected furosemide appearing in the urine un-changed. Although the plasma half-life in anephricpatients is only prolonged about three-fold (9), littleinformation is available concerning accumulation ofdrug metabolites. Nevertheless, current clinical ex-perience suggests that large doses in cases of renalfailure appear to produce only transient hypoacusia.Thus, monitoring of concentrations in plasma maybe of littleclinicalsignificance,although crucial fortoxicological and pharmacological investigations.

The main disadvantage in the use of the fluoro-metric method is that several sources of error are dif-ficult to control. The first is the presence of extracta-ble, spectrally interfering, and quenching impurities.Second, although the need to back-extract from theether phase into an aqueous medium does avoidsome adsorption phenomena on glassware and doespromote the removal of some interfering compounds,it is also a source of variability. Although within-runvariation is not great (about 5%), gross between-runvariation is an annoying source of error, which is

only partly controlled by the use of added internalstandards. In the presence of high drug concentra-tions in serum or of spectrally interfering drugs suchas salicylate, these measures are even less reliable.Fluorometry of furosemide in urine is even more dif-ficult than in serum, but the alternative (colorimet-nc) assay also has many of these same difficulties,especially adequacy of estimation of the urinaryblank value in the face of varying diuresis. Our esti-mation of the effect of diuresis as a function of urineflow is only a convenient device, and any superim-posed changes in the elimination rate of interfering

substances produced by the drug could create an un-detectable systematic error in urinary blanks by thismethod, and therefore in the derived drug concentra-tion.

In spite of these well-recognized drawbacks, use of

this assay in clinical studies of furosemide phar-macokinetics has produced reasonably accurate mea-surements of concentrations in serum and urine (10)that compare favorably to similar measurements of[35S]furosemide in normal subjects. Until other,more reliable,and convenient methods of clinicalassay are available, this method will provide a rea-sonable solution to the problem of measurement offurosemide concentration in biological fluids.

This study was supported by Hoechst Pharmaceuticals, Inc.,by contract NIH NIAMD 72-2219, and by a grant (RR-133) fromthe General Clinical Research Centers Branch, Division of Re-search Resources, NIH.

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