EUROPEAN JOURNAL OF DRUG METABOLISM AND PHARMACOKINETICS, 1979, No 3, p.143-148

A Fluorometric methodfor the determination of praziquantelin blood-plasma and urine

J. PUTTER

Institute ofPharmacokinetics, BAYER AG, wuppertal, Germany.

Received for publication: December 12, 1978.

Key-words: Praziquantel, Anthelmintic, Quantitative determination, Fluorometric method

SUMMARY

Some physicochemical data of praziquantel which may have analytical relevance are reported.For the quantitative determination praziquantel is extracted from plasma or urine by means of organic solvents and then hydrolyzed in

an aqueous alkaline solution. The hydrolyzed product is reacted with dansyl-ehloride (5-dimethylaminonaphthalene-sulfonylchloride). Thedansylated compound is separated and quantified fluorometrically.

The limit of determination is 3 p.gjl in blood plasma and 30 lJg/d in urine. For both fluids, the imprecision is approximately 7.5%.- The method is suitable for the determination ofpraziquantel in patients or healthy volunteers treated with therapeutic doses.

INTRODUCTIONPraziquantel (Embay 8440) is an anthelmintic which

may be administered to humans and which is especiallyeffective on schistosomas (1). For drugs acting againstparasites outside the intestinal tract, it is important (as isthe case with all systemically acting drugs) to know theirpharmacokinetic parameters, especially the time-courseof drug levels. Animal pharmacokinetic experiments

. with the 14C-Iabelled drug have been previouslyreported (2,3,4). For studies in humans, non-radioactivemethods are necessary. A gas-ehromatographic methodhas already been developed (5). Nevertheless, it seemedof interest to introduce, in addition, an optical method,which could be used in laboratories where gas chromato­graphic equipment is not available. A fluorometric deter­mination of the unchanged praziquantel in the blood­plasma and urine was therefore developed and is des­cribed in this report. In a future paper we will compareour fluorometric plasma values with both the gas­chromatographic values and with the radioactivity valuesobtained from the plasma after treatment with 14C-prazi­quantel (6). In another publication (7) we intend toreport on the simultaneous concentrations of praziquan­tel in the plasma and milk of lactating women. Thefluorometric method reported is very sensitive and has a

Send reprint requests to: Prof. Dr. J. PUTTER, Institute ofPharmacokinetics, BAYER AG, D 5600 Wuppertal 1, Germany.

very low limit of determination; both necessary attri­butes of a method that would be capable of quantifyingthe small concentrations occurring in the milk. - Beforeattempting to develop this method, some exploratoryexperiments on the general physicochemical propertiesof praziquantel were performed.

GENERAL PROPERTIESOF THE DRUG SUBSTANCE

The solubility of praziquantel is almost independentof the pH between pH I and pH 10. At pH 7.4 androom-temperature, it is 430 mg/l,

Stability: In neutral or weak acid or weak alkalinesolutions the concentration of the unchanged substancedid not decrease to a measurable extent (i.e. less than2%) on standing for 16 hours at room-temperature. Inthe blood-plasma, at room temperature, the contentdecreased by 5% in 16 hours. When a solution in plasmaor urine was stored at -600 C for 4 weeks, no measu­rable changes (less than 2%) were observed.

The partition coefficient (water: organic phase)between pH 1 and pH lOis practically independent ofpH. For aqueous phosphate buffer (pH 7.0): cyclo­hexane it is 14 : 86. and for the same buffer: n-hexane38: 62. In strong acids, e.g. 5- to 100normal sulfuricacid, the partition is delayed in favour of the water-

144 European Journal ofDrug Metabolism and Pharmacokinetics, 1979, No 3

phase; with 8-normal hydrochloric acid, praziquantel canbe almost quantitatively extracted from cyclohexane.The partition between alkaline solutions and organicsolvents can not be measured accurately because of theinstability of the substance in alkaline media.

Optical properties: In the ultraviolet there is anabsorption maximum at 265 nm; but the extinction of a100 mg/l aqueous solution is only 0.130. Nevertheless,this property has a certain analytical importance becauseit may be used to study the movement of the substanceduring extraction procedures and similar manipulations.- Furthermore, also unchanged Embay 8440 has a cer­tain fluorescence in a neutral aqueous solution with themaxima of 256 nm (excitation) and 294 nm (emission),but this fluorescence is extremely low compared to thatof the "dansylated" product the measurements of whichwere used as criteria for our analytical procedure.

PRINCIPLE OF THE DETERMINATIONPraziquantel (I) is extracted by means of organic

solvents from the blood-plasma or urine and cleaned byseveral washings. It is then transferred into an aqueousalkaline solution and hydrolysed producing compoundII. The hydrolysate is subjected to further washings andadjusted to a weak alkaline pH. Then it is reacted with5-dimethylarninonaphtalene-sulfocWoride{1) ("Dansyl"­chloride, III). The supposed reaction product is thecompound IV which is dansylated only once; but thepossibility of a dansylation of the other amino groupcannot be completely excluded. The excess of reagent isdestroyed and praziquantel is separated from the reac­tion mixture. Quantification was made by fluorometryand based on a comparison of the values obtained forpraziquantel and dansylamide (Y). The excitation andemission spectra of the dansylated praziquantel areshown in fig. 1 a and b. •

PROPOSED METHODGeneral remarks: It should be mentioned that the

quality of the total analysis depends to a great extent onthe purity of the glass-ware and the reagents. Foraqueous solutions, only double distilled ion-exchangedwater was used. The water must be stored in glass flasksor glassbottles and not in plastic containers. Commercialsolutions in plastic bottles (e.g. titrated acids or buffers)should not be used; it is preferable that the solution bemade in the laboratory from the solid substances orfrom the concentrated acid. Even some organic solventsof the highest available purification grade must often befurther purified by (fractionated) distillation. Errorscaused by contamination of equipment and solventscanbe recognized by the increase in the reagent blank valuesand by reduced recovery.

• Figure 1 cannot be quantitatively compared with our calibra­tion curve as the slits for excitation and emission weresmaller than those used for the calibration curve.

CXCt,-o

07,$<::CH2

"-NH-CH2-COOH TIl

Reagents (only those which require special purific­ation or are of special origin are cited) :a) Heparin-Sodium: USP XVII, 140000 U/g (Danimex,

2 Hamburg 26).b) Methylisobutylketone: A.R. (Merck, Darmstadt; Nr.

6146); purified by distillation under reduced pres­sure, followed by washing with 1/2 volume of 0.1­normal aqueous KOH and with 1/2 volume of phos­phate-buffer (pH 7.0).

c) "Dansylchloride" = 5-Dimethylarnino-naphthaline­sulfochloride-Il ) : from Merck,Darmstadt; No. 3094.For purification 1.2 g of the purchased substance isdissolved in 20 rn1 of acetone and the non-solubleportion separated by filtration. Then, after sufficientwater is added to produce a slight sedimentation, themixture is filtered a second time and the solution iscooled slowly to -2ao. The cristallized dansyl­chloride obtained after 24 hours is isolated and driedat room-temperature under vacuum.

d) "Dansylamide": for biochemical purposes (Merck,Darmstadt; No. 10852).

Procedure:

Biological material: The blood is obtained by intra­venous puncture and immediately shaken with solidheparin (0.5 to 1.0 mg per rn1 blood). The blood-cells arecentrifuged off and the supernatant plasma is kept deep­frozen until required for determination. It may be keptat - 2aoC for several days; for longer periods it shouldbe stored at -600 C. The urine is completely collectedduring the specified period, the volume is measured andan aliquot of each sample is deep frozen like the plasma.

Extraction: To 5.0 rn1 .of either the heparinizedplasma or the urine, 1.0 rn1 of 1.00normal KOH solutionis added; a pH of about 13 results. Immediately there­after, the alkalinised plasma is twice shaken with 12 rn1

J. PUtter, A fluorometric method for the determination ofpraziquantel in blood-plasma and urine 145

of a mixture of benzene and n-hexane (l : 4) each for 10min at a time. The combined organic phases are centri­fuged until clear and reduced to a volume of about 5 mlunder a continuous flow of argon at 37° C. This solutionis shaken with 0.5 ml 0.03-normal KOH solution andthen with 0.5 ml 0.03-normal hydrochloric acid. Thenthe organic phase is transferred into a small dry tube,and using a small amount of hexane the remaining dropsare rinced out of the first tube.

Hydrolysis: To the organic phase (including thewashing fluid) 1.5 ml of O.I-normal KOH are added. Theorganic solvent is completely evaporated under argon.During this process the dissolved praziquantel passes intothe aqueous phase. This phase is heated at 80° for 2hours. Then it is cooled in a water-bath at room tempe­rature. When determining praziquantel in the blood­plasma, the hydrolysate is washed with 5 rnl cyclo­hexane. When praziquantel in the urine is to be determ­ined, the hydrolysate is washed once with 5 rnl methyl­isobutyl-ketone and twice with 5 rnl each of cyclo­hexane.

Dansylation and purification: To 1 ml of the hydro­lysate, 0.45 ml of an aqueous solution containingO.I-molar phosphoric acid and 0.05-molar boric acid isadded; a pH of 9.1 results. Thereafter, 0.5 ml of adansyl-chloride solution in acetone (1.25 mmol/l) is

added. The solution is heated for 1 hour at 40° C (reac­tion with dansyl-chloride), Then the pH is adjusted toabout 13 with 0.2 rnl I-normal aqueous KOH. At thispH, the excess of dansyl-chloride is hydrolysed in 20min at room temperature. The solution is washed twotimes, using 5 rnl of cyclohexane each time. Now, byadding 0.3 rnl of a I-molar aqueous phosphoric acid, thepH of the aeueous solution is adjusted to approx. 3.From this acidified solution dansylated praziquantel isextracted with 3.0 rnl of a mixture of 9 vol. cyclohexaneand 1 vol. di-n-butylether. The organic phase is washedwith 3 rnl of O.OI-molar aqueous phosphoric acid.

Measurement: .The fluorometric measurement mustbe carried out within 1 hour after the extraction. It isperformed by use of the wave-lengths 346 om (excita­tion) and 460 om (emission). The excitation and emis­sion slits are 10 om each in the Perkin-Elmer fluoro­meter (model MPF 3). The solution to be measuredshould not be kept in the light beam for more than 2min. A solution of dansylamide, 5 x 10- 8 molar or 5 x10-7 molar, depending on the expected concentration,serves as a reference; before each series of measurementsa 5 x 10- 4 molar stock solution of dansylamide inacetone (which is stable for at least 1 month at O°C)isdiluted with the cyclohexane-butylether mixture (seeabove) to the desired concentration. This standard is

I380

I420

I

480

a

i

500i

260I

300I

340

b

Fig. 1: Spectra of the hydrolyzed and dansylated praziquantel :a) emission spectrum by excitation with 346 nm;b) emission at 460 nm using varying excitations. Slit: emission and excitation: 4 nm each.

146 European Journal ofDrug Metabolism and Pharmacokinetics, 1979, No 3

measured under the same conditions as praziquantel, andthe fluorometer is adjusted to 50 scale units.

Calculation of results: The results are calculated bypresuming a proportionality between fluorescence andconcentration (see calibration curve). From the concen­tration in the KOH solution (before hydrolysis), which isthe basis for the calibration curve, the plasma and urineconcentrations are calculated in the usual manner bycompensating concentration and dilution processes. Fur­ther, the found concentrations are multiplied by therecovery-correction factor (see below). From the calcul­ated plasma concentration the mean plasma blank valueCJ.tgJI) is subtracted. The urine concentrations are multi­plied by their corresponding volumes and the meanurinary blank value (p.gJd) is subtracted.

ANALYTICAL PARAMETERSThe determination of praziquantel seems to be an

appropriate occasion to discuss some analytical defini­tions which are of general interest in pharmacokinetic

research. Therefore, the analytical parameters of ourmethod are dealt with rather extensively.

Calibration Curve: To obtain the values for the cali­bration curve, 10 mg praziquantel are dissolvedin 50 mlacetone and diluted with O.t-normal solution of KOH tothe desired concentration.

Hydrolysis and dansylation (including the subsequentseparation procedures) are carried out as describedabove. Thus, the calibration curve in Fig. 2 refers to theconcentration in the KOH solution before hydrolysis(i.e. approximately the threefold value of the plasmaconcentration and of the theoretical molar concentra­tion in the cuvette). - Although for determination inthe urine, the procedure after hydrolysis was slightlychanged, no significant change in the calibration curvewas observed.

A concentration of 5 x 10- 8 mol dansylamide per 1(in the cuvette) is equal in fluorescence to 131 ngpraziquantel per ml aqueous KOH. The calibration curvepasses through the zero-point of the coordinates andrepresents a straight line from 25 ng to 2 p.g per ml

100J500

Ooncentranon [ng/ml]

20010050

50

100

Fluorescence[Scale units]

200

500

Fig. 2: Calibration curve of praziquantel in a log-log. plot in comparison to dansyl amide as standard. The higher values (above 100, i.e. thelimit of the scale) are measured with one tenth of the usual fluorometer sensitivity and then multiplied be ten.

J. PUtter, A fluorometric method for the determination ofpraziquantel in blood-plasmaand urine 147

Recovery(%)74.272.777.5

(provided an appropriate adjustement of the sensitivityof the fluorometer is made). Therefore, in the doublelogarithmic plot it represents a straight line with a slopeof 45°.

With concentrations below 25 ng/ml, no deviationfrom proportionality could be detected.

Recovery yield: The yield of the chemical reaction(hydrolysis and dansylation including the subsequentwashings and extractions) cannot be determined becausewe do not know the fluorescence of the pure dansylatedpraziquantel. However the small deviations from astraightlined calibration curve demonstrate that this partof the overall recovery is a rather constant proportion.In comparison to an equimolar solution of dansylamidethe molar fluorescence of the dansylation product is35%. However, it does not seem justified to concludethat the yield of the chemical reaction is only 35%,because it is not known to what extent the rest of themolecule influences the intensity of fluorescence.

Based on these considerations, the term "recovery"used in this publication refers to the percentage ofsubstance obtained from the biological material (plasmaor urine) including all steps up to the onset of thechemical reaction, i.e. the hydrolysis. Possible furtherlosses need not be considered in the calculation becausethey are contained in the calibration curve.

To 16 plasma samples taken from different persons atvarious times of the day (and at various times after themeals) we added 40 ng per ml plasma and determinedthe concentration as described. After subtraction of theblank values (see below) the mean recovery was 31.2 ng/rnl± 2.24 ng/ml (standard deviation) or 78.0% ± 5.6%.Therefore, the plasma levels found after administrationin vivo have to be multiplied by the correction factor1.28.

To find a possible concentration dependency of therecovery, we determined, as the mean of 3 determin­ations each, the recoveries of different concentrationsadded to same plasma (pooled plasma from 10 persons).The following values demonstrate that the recovery(within the margin of error) was independent of theconcentration in the plasma:

Concentration(ng/ml)

2004020

To establish the recovery from urine, 24 hour urinesamples obtained from 10 persons were added with 60ng praziquantel per ml. The mean recovery was 84.4% ±6.5% (standard deviation). Thus the correction factor is1.18.

Imprecision: The imprecision is defmed by the Inter­national Federation of Clinical Chemistry (8) * as therelative or percentage standard deviation from the meanvalue. As the above-mentioned standard deviations ofrecovery comprises all experimental errors includingthose which are based on the imprecision of the calibra­tion curve, this standard deviation is a mesure of theimprecision of the whole determination. Thus the impre­cision for the plasma is 1.28 x 5.6% = 7.2% and for theurine 1.18 x 6.5% = 7.7%.

Blank values: The reagent blank value (obtained byperforming all procedures without adding biologicalmaterial) calculated as the mean of 10 determinationscorresponded to a plasma-concentration (after multi­plying by the correction factor) of9 ng/ml + 1.2 ng/ml.

Plasma blank value: 13 plasma samples of differentorigin (see above) were treated in the same manner asdescribed for the determination of praziquantel. Theresulting fluorescence was calculated as apparent prazi­quantel after subtraction of the reagent blank and multi­plying by the recovery-correction factor. Thus the meanbiological blank value was found to be 0.8 ng/ml ±1.4 ng/ml. (The relatively high variation of the biologicalblank is due mainly to the variation of the reagentblank).

Urinary blank value: 10 samples of urines collectedduring 24 hours from untreated healthy subjects wereprocessed as for the determination of praziquantel. Asdescribed for the plasma, the apparent praziquantel con­centrations were calculated and then multiplied by thecorresponding 24-hour volume values **. This apparentdaily excretion of praziquantel was 23.9p.gjd with astandard deviation of ± 13 p.g/d.

The limit of determination (detection limit) is thesmallest single result which can be distinguished with adegree of probability from the biological blank value(8,9). If we assume that the observed proportionality isapplies also so with low values and that the biologicalblanks follow a normal distribution, the limit may becalculated statistically by using the variation of the bio­logical blank values. On the basis of a 95% tolerance (5%probability for false positive results), the detection limitaccording to this method would be 2.26 x 1.4 ng/ml ::::::3 ng per ml plasma or 2.37 x 13 p.g/d :::::: 30 p.g/d in theurine.

Specificity : The mean biological blank value was sosmall that it did not differ significantly from that of the

• The percentage standard deviation is often called "variationcoefficient". Because it does not represent a coefficient inthe real sense of the word, the use of the term "variationcoefficient" should be avoided.

•• As for urine, it seems more reasonable to define the limit ofdetermination as an amount of substance divided by thecollection time (mostly 1 day) rather than as a concentration;for the blank values calculated for a given time do not vary asmuch as those obtained for the different concentrations.

148 European Journal ofDrug Metabolism and Pharmacokinetics, 1979, No 3

reagent blank. It can be assumed, therefore, that in theplasma of healthy subjects, there are no substanceswhich could simulate praziquantel. In extracts from theplasma of praziquantel-treated subjects, we found thesame excitation and emission spectra as for dansylatedpraziquanteI. This justifies the assumption that with ourmethod, only unchanged praziquantel is determined.Proof of this assumption is still lacking, however, asduring hydrolysis a part of the molecule is split off andthe possibility of a metabolic step in this part cannot beexcluded. Since the chemical reaction upon which thequantification of praziquantel is based could also beapplicable to the metabolites of this product, the valid­ity of our technique cannot be definitively establisheduntil all of the metabolites have been determined.

DISCUSSION

The method described in this paper has already beenapplied to the quantification of praziquantel in plasmaof humans who had received therapeutic doses of theproduct. The concentrations of the product in theplasma were readily measurable and it was found thatmost of it was in the form of unchanged product. It isnot yet established whether this is also the case in urine.

Our quantitative determination was primarily deve­loped under the assumption that a highly sensitivemethod with a very low limit of determination would benecessary. It was found, however, that praziquantel,being well tolerated, can be given to humans in higherdoses than was originally assumed. Nevertheless, thesmall limit of determination has an advantage in that thekinetics can be followed down to very low concentra­tions. Furthermore, for certain indications, very smalldoses are sufficient and in these cases, the maximumblood-level amounts to at least the lO-fold volume ofour determination limit. In another paper we shalldemonstrate that praziquantel is excreted in the milk invery small amounts and that the course of this excretioncan be determined only by means of a sensitivemethod.An analytical parameter we intentionally did not men­tion is sensitivity. This parameter is only vaguely definedin the afore-mentioned guidelines (8,9) and it seemsreasonable not to use this term until a more precise andquantifiable definition is established. Furthermore, thecited definition (8,9) does not give sufficient informa­tion.

The imprecision of the results obtained when quanti­fying small amounts as well as the variation of thebiological blank values are mainly due to the variation ofthe reagent blanks. These depend partly on certain inevi­table impurities of the glass-ware and of the solvents andpartly to byproducts of the excess dansylchloride. Forsafety reasons we chose rather small plasma­concentrations when determining the imprecision. Itshould therefore be mentioned that with higher plasma­concentrations, the results would be influenced only to aslight extent by the variation of the blanks, and theimprecision would probably be smaller than indicated inthe present study.

REFERENCES

1. Thomas H. and Gonnert R. (1977) : The efficacy of prazi­quantel against cestodes in animals, Z. Parasitenkunde, 52,117-127.

2. Steiner K., Garbe A., Diekmann H.W.and Nowak H. (1976) :The fate of praziquantel in the organism. I. Pharmacokineticsin animals, Europ. J. Drug Metab. Pharmacokin., 1,85-95.

3. Steiner K. and Garbe A. (1976) : The fate of praziquantel inthe organism. II. Distribution in rats, Europ. J. Drug Metab.Pbarmacokin.,1 ,97-106.

4. Diekmann H.W. and Biihring K.O. (1976) : The fate of prazi­quantel in the organism. III. Metabolism in rat, beagle dogand rhesus monkey, Europ. J. Drug Metab. Pharmacokin., 1,107-112.

5. Diekmann H.W. (1979) : Qunatitative determination of Prazi­quantel in body fluids by gaz liquid chromatography. Europ.J. Drug Metab. Pharmacokin., 4,139-141.

6. Patzschke K., PUtter J., Wegner L.A., Horster F.A.,Diekmann H.W. (1979): Serum concentrations and renalexcretion in humans after oral administration of Praziquantel- results of three determination methods - Europ. J. DrugMetab. Pharmacokin., 4, 149-156.

7. PUtter J., Held F. (1979) : Content of praziquantel in blood­plasma and milk of lactating women, to be published in thisjournal.

8. BUttner J., Borth R., Boutwell J.H. and Broughton P.M.G.(1975) : Provisional Recommendation on quality control inclinical chemistry; Part 1. General principles ans terminology,Z. Klin. Chern. Klin. Biochem., 13, 523-531.

9. Biittner J., Borth R., Boutwell J.H., Broughton P.M.G. andBowyer R.C. (1976) : Provisional recommendation on qua­lity control in clinical chemistry; Part 2. Assessment of analy­tical methods for routine use. Z. Klin, Chern. KIin. Biochem.,14,265-275.