Volume 13 Issue 7 Analytical CHEMISTRY - · PDF filechromatography tandem mass spectrometry Full Paper ... polythiazide, eplerenone, 2-amino-4-chloro-1,3-benzenedisulphonamide

Comprehensive screening of diuretics in human urine using liquidchromatography tandem mass spectrometry

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INTRODUCTION

Diuretics are therapeutic agents that are used toincrease the rate of urine flow and sodium excretion inorder to adjust the volume and composition of bodyfluids or to eliminate excess of fluids from tissues Theyare used in clinical therapy for the treatment of variousdiseases and syndromes including hypertension heartfailure liver cirrhosis renal failure kidney and lung dis-eases[1] Diuretics were first banned in sport (bothincompetition and out of competition) in 1988 becausethey can be used by athletes for two primary reasons

Shobha Ahi1 Alka Beotra1 GBKSPrasad2

1National Dope Testing Laboratory Ministry of Youth Affairs and Sports CGO Complex Lodhi RoadNew Delhi-110003 (INDIA)

2SOS in Biochemistry Jiwaji University Gwalior (INDIA)E-mail drabeotragmailcom

First their potent ability to remove water from the bodywhich can cause a rapid weight loss that can be re-quired to meet a weight category in sporting eventsSecond they can be used to mask the administration ofother doping agents by reducing their concentration inurine primarily because of an increase in urine volumeThe urine dilution effect of diuretics also allows them tobe classified as masking agents and precludes their useboth in and out of competition Some diuretics also causea masking effect by altering the urinary pH and inhibit-ing the passive excretion of acidic and basic drugs inurine[2-4]

KEYWORDS

Doping diureticsLC-MSMS WADA

Drugs of abuse

ABSTRACT

Diuretics are drugs that increase the rate of urine flow and sodium excretionto adjust the volume and composition of body fluids There are severalmajor categories of this drug class and the compounds vary greatly instructure physicochemical properties effects on urinary composition andrenal haemodynamics and site mechanism of action Diuretics are oftenabused by athletes to excrete water for rapid weight loss and to mask thepresence of other banned substances Because of their abuse by athletesdiuretics have been included in the World Anti-Doping Agencys (WADA)

list of prohibited substances The diuretics are routinely screened by anti-doping laboratories as the use of diuretics is banned both in-competitionand out-of-competition This work provides an improved fast and selectiveliquid chromatographytandem mass spectrometric (LCMSMS) method

for the screening of 22 diuretics and probenecid in human urine The samplespreparation was performed by liquid-liquid extraction The limit of detection(LOD) for all substances was between 10-20 ngml or better The methodwas successfully applied to 21916 routine doping control samples 2013 Trade Science Inc - INDIA

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Volume 13 Issue 7

Analytical CHEMISTRYAnalytical CHEMISTRYISSN 0974-7419

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Shobha Ahi et al 271

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Although the main application of diuretics is to en-hance renal excretion of salt and water their effects arenot limited to sodium and chloride they may also influ-ence the renal absorption and excretion of other cat-ions (K+ H+ Ca

2+ andMg

2+) anions (Cl- HCO

3- and

4-) and uric acid This pharmacological class of

drugs includes compounds with a variety of pharmaco-logical and physicochemical properties Because of thevariety of diuretic compounds classification of thesedrugs can be based on different criteria The most com-mon classification categories are by site of action in thenephron relative efficacy chemical structure effects onpotassium excretion similarity to other diuretics andmechanism of action[1]

There are several classes of diuretic drugs basedon their mechanism of actionThiazides (eg

benzthiazide) loop diuretics (eg bumetanide) potas-sium sparing diuretics (eg amiloride) carbonic anhy-

drase inhibitors (eg acetazolamide) osmotic diuret-ics (eg mannitol)and mercurial diuretics (eg mersa-lyl) (Figure 1)

Diuretics are relatively polar hence are amenableto analysis by high performance liquid chromatography(HPLC) using C18 type phases with diode array (DAD)or fluorescence detection[56] However whilst thismethod may be suitable for screening purposes it suf-fers from significant interferences due to the backgroundoccurring in urine samples The information providedby HPLC is insufficient for confirmation as alternativemass spectral confirmation of identity is needed fordoping control To overcome this problem detectionof diuretics by gas chromatography mass spectrometry(GC-MS) was introduced In order to improve thevolatility of the diuretics either methylation or silylationwas performed prior to GCMS The methylation of

these polar drugs has been the most suitable proce-

Figure 1 Site and mechanism of action of diuretics (A) The nephron with major divisions labeled (B) Mechanism ofcarbonic anhydrase inhibitors in the proximal tubule (C) Mechanism of the Na+K+2Cl- symporter inhibitors in the thickascending limb of the loop of Henle (D) Mechanism of the Na+Cl- symporter inhibitors in the distal tubule (E) Mechanismof renal epithelial Na+ channel inhibitors and mineralocorticoid receptor antagonists in the collecting duct Aldo aldosteroneCA carbonic anhydrase MR mineralocorticoid receptor Figure modified from Jackson (2006)

272 Comprehensive screening of diuretics in human urine

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ACAIJ 13(7) 2013

dure[7-9] and has allowed GCMS to be used as a screen-

ing and confirmatory method by replacing the less se-lective HPLC procedures

Later a rapid method was published using micro-waves to assist the methylation after extraction[10] Fewproblems encountered with the methylation procedureprior to GCMS approach were the difficulty in me-

thylating some diuretics and the toxicity of the methyliodide used in the derivatization process With the ad-vent of liquid chromatography tandem mass spectrom-etry (LCMSMS) instrumentation a technique par-

ticularly suited to the detection of polar substances theopportunity has arisen to develop methods for the de-tection and confirmation of diuretics without the needfor derivatization[11-13]

The aim of this work was to develop a fast andsimple LCMSMS method for the detection of diuretics

and probenecid that could replace the existing GCMS method based on derivatisation following methyla-tion The method was also required to reliably detectadditional diuretics which could not be detected by ex-isting method

EXPERIMENTAL

Chemicals and reagents

All reagents were analytical grade or HPLC gradeAcetonitrile and ethyl acetate were purchased fromQualigens Mumbai India tertiary butyl methyl etherand formic acid 98 were purchased from MerckMumbai India HPLC mobile phases were filteredthrough a 02 microm PTFE filter Ultra high purity nitrogen

was obtained from nitrogen generator plant installed atthe laboratory Standards of the following diuretics wereobtained from reliable sources acetazolamidehydrochlorthiazide chlorthiazide chlorthalidoneindapamide furosemide bumetanidebendroflumethiazide ethacrynic acid probenecidmefruside canrenone spironolactone triametrene andamiloride (SigmaAldrich USA) and benzthiazide

cyclopenthiazide cyclothiazide epithiazidehydroflumethiazide polythiazide eplerenone 2-amino-4-chloro-13-benzenedisulphonamide (ACB) 4 amino-6-trifluoromethyl-benzene-13-disulphonamide (ATFB)(courtesy of the World Association of Anti-Doping Sci-entists-WAADS) and metolazone (courtesy of the In-

stitute of Biochemistry Cologne Germany) Water waspurified using a Milli-Q water purification system in-stalled in the laboratory (Millipore Bedford USA)

Sample preparation

The sample preparation was performed using thepre-set method for extraction of various categories ofdrugs in the laboratory involving enzymatic hydrolysisfollowed by liquid-liquid extraction[14] To two or fourml of urine sample aliquots (based on specific gravity)250 ngml of internal standard (mefruside) was addedThe urine samples were hydrolysed by acirc glucuronidase

(Ecoli) enzyme at 60degC for an hour at pH 70 using

02 M phosphate buffer The pH was adjusted to 9-10with 7 K

3 and liquid-liquid extraction was per-

formed using 5 ml TBME After mixing for 15 minutesand centrifugation for 10 minutes at 3000 rpm the or-ganic layer was separated The pH of the aqueous layerwas adjusted to 2-3 by 6 N HCL and second extrac-tion was done using 4 ml ethyl acetate After mixing for15 minutes and centrifugation for 10 minutes at 3000rpm the organic layer was mixed with the first one andevaporated under nitrogen gas at 60degC Finally the resi-

due was reconstituted in 100 igravel of mobile phase (1Formic acid and Acetonitrile) (5050) (vv) and trans-ferred into conical autosampler vials for analysis

Instrumentation

Negative polarity

A Waters Aquity ultra performance liquid chroma-tography (UPLC) separation module equipped with abinary pump and C18 column (Acquity BEH17microX21X100mm) was used for the LC separation

The mass spectrometer used was API 4000 QTrap triplestage quadrupole platform The following binary mo-bile phase gradient was formed by solvent A (1 aque-ous formic acid) and solvent B (acetonitrile) at a flowrate of 03mlmin 40 B to 80 B in 400 min andthen 40 B in 500 min The injection volume was 5 microl

The spray conditions of the API interface were per-formed under Electro Spray Ionization (ESI) where theinterface temperature was 450ordmC and IS voltage was4500 Volts

Positive polarity

An Agilent 1100 series high-pressure gradientpumping system and autosampler (Agilent Technolo-

gies Waldbronn Germany) and an API 3200 TM tan-dem mass spectrometer (AB Sciex Canada) operat-ing with an electrospray ionization source A C18 col-umn (Intersil- C-18 ODS-3 (30 mm 50mm x 46mm)was used The mobile phase delivered at a flow rate of07 mlmin consisted of Solvent A (1 formic acid inwater) and Solvent B (acetonitrile) The gradient pro-gram was 0min 15 B 400 min 60 B 700 min

100 B 11 min 15 B The injection volume was

10 microl The mass spectrometer operating conditions con-

sisted of a source heater probe of 550 ordmC with aTurboIonspray voltage of 5500 V entrance potentialof 10 curtain gas setting of 10 and CAD setting of 4The compound dependent parameters were optimizedfor each compound and nitrogen was used as the colli-sion gas Collision energies were different for differentanalytes and are listed in TABLE 1

Preparation of reference solutions and quality con-trol samples

A stock standard solution was prepared of eachindividual compound at a concentration of 1 mgml inethanol and stored at -20 ordmC The reference working

solution mix of the compounds was prepared at theconcentration level of 10 microgml in ethanol Urinary quality

control samples were prepared with every batch at aconcentration level of 200 ngml

Method development and validation

The analytical method was developed and validatedas per the WADA guidelines for the anti-doping labo-ratories[14] For validation the parameters specificity ionsuppression intra and inter-day precision limit of de-tection (LOD) and robustness were determined

Recovery

The recoveries of the diuretics excreted as parentcompounds were determined by spiking five replicatesof blank urine with each analyte at a concentration of200 ngml and comparing these results with anunextracted standard

Specificity

Evaluation of specificity was carried out by analyz-ing six different spiked and six different blank urinesamples collected from healthy volunteers to test forinterfering signals in the selected MRM chromatograms

at expected retention times of the analytes

Ion suppressionion enhancement

The extent of ion suppression or enhancement wasinvestigated by analysing six different blank urinesamples via post-column continuous infusion of a mix-ture of the reference compounds (1 mgml 20 mlmin)[15]

Precision

Intra-day precision was determined at 200 ngmlfor each compound using five replicates of spiked urinesamples The corresponding inter-assay precision wascalculated from samples prepared and analyzed at threedifferent days (n=5day) The precision of the methodwas determined by calculation of the coefficient of varia-tion (CV) of the area ratio of the ion transition of theanalytes and the internal standard

Limit of detection (LOD)

The LOD was defined as the lowest concentrationof analyte that can be identified measured and reportedIt was calculated using two diagnostic ions with a sig-nal-to-noise ratio greater than 3 The LOD was esti-mated via signal to noise ratio (SN) of the respectiveion traces using ten blank samples and ten fortifiedsamples at concentration levels from 10 to 50 ngml

Applicability to excretion study samplesroutinedoping control samples

A total of 21916 doping control samples receivedin National Dope Testing Laboratory (NDTL) Indiafrom 2008 to 2012 were analyzed by the developedmethod for diuretics and probenecid The samples ofmajor events viz III Commonwealth Youth Games ISingapore Youth Olympic Games and XIX Common-wealth Games were also included in the study Themethod was also applied to excretion study samplesafter oral administration of spironolactone andeplerenone to human volunteers The study was dulyapproved by the ethics committee of NDTL India

RESULTS

A total of 22 diuretics and the masking agentprobenecid were detected by the method (Figure 2)As the certified reference compounds for all the diuret-

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Figure 2 Chemical structure of following diuretics (a) Acetazolamide (b) Amiloride (c) Bendroflumethiazide (d)Benzthiazide (e) Bumetanide (f) Canrenone (g) Chlorthiazide (h) Chlorthalidone (i) Cyclopenthiazide (j) cyclothiazide(k) Etacrynic acid (l) Epithiazide (m) Eplerenone (n) Furosemide (o) Hydrochlorthiazide (p) Hydroflumethiazide (q)Indapamide (r) Metolazone (s) Polythiazide (t) Spironolactone u) Triametrene (v) Probenecid (w) ACB (x) ATFB

ics and probenecid were available direct infusion analy-sis was opted to obtain instrumental conditions andreference product ion spectra

Both positive and negative modes of ionization wereused for each analyte to obtain mass spectra (TABLE1) The MSMS data were obtained by choosing a

precursor ion and measuring the product ion transitionsfor each compound for both modes of ionization Themost appropriate precursor to product ion transition ineach ionization mode was selected for each compoundand this was optimized to obtain maximum sensitivityand specificity The chromatographic run was optimizedtaking into account the chemical versatility of theanalytes resulting in a wide range of polarities Thedetection of 22 diuretics and probenecid was accom-plished While 4 analytes were detected in positive ionmode as protonated quasi-molecular ions [M+ H]+ 19analytes were detected in negative ionmode asdeprotonated quasi-molecular ions [M-H]- All analyteswere clearly detectable at required concentration lev-

els (Figure 3) No interfering signals of the matrix weredetected at the expected retention times of the analytesproving the specificity of the method

Stable retention times are of utmost importance forreliable evaluation Analysis of QC samples over fourweeks yielded stable retention times (CV lt 2) for allof the compounds No significant decrease or increaseof the electrospray response at the expected retentiontimes of the analytes was observed when the urinarymatrix was injected disproving the phenomenon of ionsuppression or enhancement in the developed method

The LOD of different compounds in the developedmethod is listed in TABLE 2 The linearity was evalu-ated from 10 to 200 ngml in urine for all parent druganalytes All gave a linear response with correlationcoefficients (R2) ranging from 0975 to 0994 The re-covery percentage for all the analytes was found to bebetween 887-110 intra-and inter-day precisionsshowed coefficients of variation less than 15 for allanalytes (TABLE 2)

The method was successfully applied to the analy-sis of 21916 routine samples received in NDTL from2008 to 2012 A total of 508 adverse analyticalfindings (AAFs) for various drugs of abuse were re-ported during the period (Figure 4) Out of the totaladverse analytical findings 647 of AAFs were con-stituted for diuretics (Figure 5) In 2009 a steep rise inAAFs of diuretics was observed after the inclusion ofmethod in the routine screening procedure which maybe due to the improved method The drug-wise breakupshows that most abused diuretic was furosemide (Fig-ure 6)

Eplerenone is methyl hydrogen 911 aacute-epoxy-17 aacute-hydroxy-3-oxopregn-4-ene-7 aacute21-dicarboxylateatilde-lactone and ahighly selective aldosterone blockerandpotassium-sparing diuretic used in the therapy of hy-pertension The excretion study samples of eplerenonewere collected for 100 hours after oral administration

of single dose of Eptus 25 mg (Glenmark India) to 3healthy male volunteers (age-25plusmn2 years) The excre-

tion study rate of the parent compound was quantitatedin urine using a five level calibration curve with r=09987 The highest concentration of the drug was foundat 6 hours (Figure 7) Additionally the metabolite 6 acirc-OH eplerenone was also monitored and was seen till75 hours post administration (Figure 8) The precursorto product ion transitions of mz 415-163 and mz 431-337 were used to monitor eplerenone and 6 acirc-OH

eplerenone respectively Acceptable precision and ac-curacy were obtained for concentrations over the stan-dard curve range

Spironolactone is a mineralocorticoid receptor an-tagonist (aldosterone antagonist) and a potassium-spar-ing diuretic The excretion study samples of spirono-lactone were collected for 100 hours after oral admin-istration of single dose of Lasilactone 20 mg (Aventis

TABLE 1 Mass spectrometric parameters

Compound Ionization

mode Molecular

weight Precursor Ion (mz)

Product Ion (mz)

Collision Energy

Declustering potential (V)

1 Acetazolamide - 22225 221 83 142 -40 -35 -40 -40

2 Amiloride + 2296 230 171 116 36 43 39 39

3 Bendroflumethiazide - 4214 420 289 328 -41 -36 -40 -40

4 Benzthiazide - 43194 430 228 308 -38 -35 -48 -48

5 Bumetanide - 36442 363 319 80 -20 -40 -40 -40

6 Canrenone - 34046 341 187 107 47 51 48 48

7 Chlorthiazide - 2957 294 214 179 -30 -42 -40 -40

8 Chlorthalidone - 3387 337 190 146 -41 -40 -35 -35

9 Cyclopenthiazide - 37991 378 205 269 -39 -36 -44 -44

10 Etacrynic acid - 30313 301 243 207 -38 -43 -40 -40

11 Epithiazide - 425 424 268 404 -39 -18 -41 -41

12 Eplerenone - 41450 415 163 337 48 39 51 51

13 Furosemide - 3307 329 285 205 -30 -35 -40 -40

14 Hydrochlorthiazide - 2977 296 269 -25 -40

15 Hydroflumethiazide - 3312 330 239 302 -33 -21 -39 -39

16 Indapamide - 365 364 189 132 -40 -45 -40 -40

17 Metolazone - 3658 366 259 277 34 48 47 47

18 Polythiazide - 439 438 324 418 -38 -32 -43 -43

19 Spironolactone + 4165 314 187 107 47 51 48 48

20 Probenecid + 2853 286 244 185 25 38 33 33

21 Triamterene + 2532 254 237 195 25 38 36 36

22 ACB - 28573 284 207 169 -25 -38 -30 -30

23 ATFB - 31928 318 214 239 -38 -32 -34 -34 - negative + positive

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Figure 3 Detectability of the method showing ion chromatogram of all analytes

Pharma Ltd India) to 3 healthy male volunteers (age-25plusmn2 years) During electrospray evaporation and ion-ization process spironolactone readily loses the 7aacute-acetylthiogroup being transformed to canrenone addi-

tionally it is readily metabolised in humans to canrenoneso it was quantified in that form The excretion studyrate was quantitated in urine using a five level calibra-tion curve with r = 09971 The highest concentration

of canrenone was found at 12 hours (Figure 9) Theprecursor to product ion transitions of mz 341-187was used to monitor canrenone in urine samples Ac-ceptable precision and accuracy were obtained for con-centrations over the standard curve range

DISCUSSION

As previously mentioned diuretics are commonlyprescribed in clinical medicine to treat hypertension andother cardiovascular disorders Diuretics are banned inall sports because they can cause rapid weight loss andcan act as masking agents (to hide the effects of otherprohibited substances) both in and out of competitionHowever the World Anti-Doping Code permits thetherapeutic use of diuretics when athletes and their phy-sicians apply for therapeutic use exemptions (TUE)according to the International Standard for TUE[28] Fordiuretics the primary permitted therapeutic use is forhypertension It should be noted that a TUE is not validif an athletes urine contains a diuretic in associationwith a threshold or sub-threshold level of another ex-ogenous substance included on the Prohibited List

Reasonably the most effective use of diuretics insport doping would be before an anti-doping test Di-uretics increase urine volume and dilute any dopingagents as well as their metabolites present in the urineand make their detection more problematic by conven-tional anti-doping analysis For this reason diuretics areclassified as masking agents on the WADA ProhibitedList (class S5 Diuretics and other masking agents)[4]

For the detection of diuretics in urine in sports dop-ing a single minimum required performance level(MRPL) of 200 ngml is fixed by WADA for accred-ited laboratories[17] Even though the relative potenciesmetabolism and elimination properties vary dramaticallyand result in different urinary levels between the classesof diuretics (TABLE 3) the MRPL at 200 ngml is suf-ficient to detect acute diuretic abuse by athletes Lowerdosages of diuretics are likely to be insufficient at caus-ing the masking effect or dramatic and acute weightloss abusers seek

Several analytical techniques have been proposedfor the analysis in doping control primarily among themare HPLC-UV-DAD GC-MS LC-MS and LC-MSMS[17-26] However the best solution for a com-

prehensive screening method capable of detecting thepresence in a biological sample at the same time satis-fying the WADAs MRPL is represented by the meth-ods based on LC-MS or LC-MSMS[1-131819]

When diuretics were introduced on the list of for-bidden substances by the International Sports Authori-ties the first attempts to create a screening method fortheir detection were based on HPLC with UV diodearray as detector as it facilitated peak identification[56]According to WADA the confirmation procedure datato support a positive case should be based on massspectrometry This made the use of GC-MS a methodof choice[15] As a matter of fact GC-MS is still usedby various anti-doping laboratories for the detection ofdiuretics The analytical procedure based on GC-MSis structured into the series of following events samplepre-treatment chemical derivatization and injection onGC-MS Derivatization is necessary prior to GC-MSanalysis as most of the diuretics are not sufficiently vola-tile lipophillic or thermally stable to be directly assayedwith the analytical technique The most commonderivatization procedures are silylation and methylationbut the latter is preferred as it allows sufficient yields ofmore stable derivatives[30] However the method facessome pitfalls because of the tedious and hazardousmethylation step and not-so-sensitive GC-MS analysisto give lower limit of detection (LOD)

The attempts to use LC-MS (MS) for the detec-tion of diuretics started in early 1990s In the past de-cade LC-MSMS has proven to be the best methodof choice for the analysis of diuretics for the followingreasons first it does not require the chemicalderivatization of the samples prior to the analysis andsecond it does not require the sample to be convertedinto gaseous phase before thereby improving sensitivityand lowering the LOD The use of simultaneous posi-tive and negative ionization method allowed the detec-tion of acidic as well as basic diuretics The analysiswith tandem MS with triple stage quadruples proved tobe highly sensitive and specific The improvements inthe scanning speed of the mass spectrometers as wellas better performing LC columns and LC pumps(UPLC) allowed an increase in speed of analysis[11-

131819]Due to the compatibility of the LC-MSMS system

with the aqueous matrix of urine and high sensitivity the

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ACAIJ 13(7) 2013

identification of prohibited substances without pre-con-centration steps is possible resulting in very simple andfast dilute-and-shoot methods Additionally omission

of sample pre-treatment steps leads to savings in termsof labour and reagent costs error-prone and time Di-uretics are excreted as parent compound in humanshence are suitable candidates for direct dilute andshoot urine analysis Various direct urine analysis meth-ods for diuretics were developed in the past[2829] How-ever the dilute and shoot approach possess few pitfallslike shifted retention times due to unwanted matrix ef-fects frequent degradation of LC column contamina-tion of source and analysers of the mass spectrometeretc Hence the dilute and shoot methods may be a goodchoice for confirmatory procedures but they could notbe adapted for routine screening purposes

The members of diuretic class vary greatly in termsof structure physicochemical properties and site ofmechanism of action In the 1990s the analysis of di-uretics in doping by LC-UV and GC-MS methods wasa challenge for the anti-doping laboratories due the het-erogeneity of the substances included Since the ad-

Figure 4 Total number of samples and AAFs during 2008-2012

Compound LOD

(ngml) Recovery

() RSD ()

RRT- precision

Inter-day Precision (CV)

(n=5X3)

Intra-day precision

(CV) (n=5) 1 Acetazolamide 20 924 109 012 23 32

2 Amiloride 20 887 71 021 45 41

3 Bendroflumethiazide 10 985 142 012 56 48

4 Benzthiazide 10 1018 124 08 12 2

5 Bumetanide 10 1084 132 023 22 21

6 Canrenone 10 945 78 024 48 41

7 Chlorthiazide 20 925 89 015 59 49

8 Chlorthalidone 20 874 98 022 55 44

9 Cyclopenthiazide 20 981 76 09 39 32

10 Etacrynic acid 20 874 74 015 28 25

11 Epithiazide 20 956 94 013 33 31

12 Eplerenone 10 993 101 023 34 28

13 Furosemide 10 1052 12 012 48 4

14 Hydrochlorthiazide 10 942 118 012 58 52

15 Hydroflumethiazide 20 96 112 013 68 65

16 Indapamide 10 1045 85 009 52 5

17 Metolazone 10 110 94 013 44 5

18 Polythiazide 20 947 125 014 25 38

19 Spironolactone 10 987 13 017 35 32

20 Probenecid 10 947 95 026 44 43

21 Triametrene 10 895 89 017 55 52

22 ACB 15 781 77 01 48 44

23 ATFB 15 812 42 012 39 41

TABLE 2 Method validation results showing recovery and precision

Figure 6 Drug wise distribution of AAFs for diuretics in NDTL (2008-2012)

Figure 5 Year wise distribution of AAFs for diuretics in NDTL (2008-2012)

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Figure 8 Urinary excretion profile of 6 acirc-OH eplerenone after oral administration

vent of robust and reliable LC-MSMS instrumenta-tion their detection in human urine is no longer a prob-lem This has increased the sensitivity of the methodand the number of compounds in the screening proce-dure whereas decreased the analysis time and cost tothe laboratories

The method described in this study has confirmedthat both positive and negative ionization are required

for detection of diuretics on LC-MSMS For the com-pounds included in the study 04 were detected in posi-tive ionization mode while 19 were included in the nega-tive ionization mode The use of solvent systems usingwater with added ammonium acetate formic acid andacetic acid combined with methanol or acetonitrile wasevaluated The best combination of ionisation efficiencyand chromatographic peak shape was found with the 1

Figure 7 Urinary excretion profile of eplerenone after oral administration

Figure 9 Urinary excretion profile of spironolactonecanrenone after oral administration

formic acid acetonitrile combination Until the de-velopment of this method diuretics were screened andconfirmed in NDTL India using GCMS methylation

method However screening of diuretics was shifted toLC-MSMS after full and successful method develop-ment and validation All the compounds were found tohave minimum three product ions of sufficient intensityto enable effective confirmation by comparing their ionratios

The current screening method on LC-MSMS takesonly 5 min of runtime to analyze 1 sample against the15 min runtime of the traditional GC-MS method Thishas significantly improved the throughput where 12samples could be detected in an hour against 4 samplesper hour using the GC-MS method The high-through-put of the method proved to be beneficial during thetesting of major events viz I Singapore Youth OlympicGames and XIX Commonwealth games

The method developed has shown itself to be simplerobust and reliable in the detection of diuretics for sportsdrug testing It has been in routine use for more than 5years involving the analysis of over 20000 urine samplesOnly four UPLC columns were needed for this periodUse of the GCMS diuretics screening proceeding had

been discontinued after running both methods in paral-lel for 1 month A rise in AAFs for diuretics was ob-

served in 2009 after employing the improved methodin routine testing procedure Maintenance of the API4000 Qtrap has been minimal merely involving occa-sional washing of the curtain plate to restore sensitivityApart from the intended advantages of screening di-uretics with faster simpler and safer sample prepara-tion it has also been found that the interpretation of thedata is much easier as interfering peaks are very rarelyfound in samples

CONCLUSION

A fast generic and sensitive method was devel-oped for the analysis of 22 diuretics and probenecidThe experiments presented in this work were based onultra-high-pressure liquid chromatography coupled tohybrid quadruple tandem mass spectrometry Themethod was validated according to the InternationalStandard for Laboratories described in the World Anti-Doping Code and was selective enough to comply withthe World Anti-Doping Agency recommendations

ACKNOWLEDGMENT

The support of Ministry of Youth Affairs and Sportsis duly acknowledged for financial grant

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TABLE 3 Properties of diuretics important for analytical method development

Compound pKa logP Relative potency

Elimination route

Metabolism

Category

1 Acetazolamide 74 91

03 1 Renal 0 Carbonic anhydrous inhibitor

2 Bumetanide 36 77

26 40 Renal 38 Hepatic

3 Furosemide 38 75

20 1 l Renal 35 Renal

4 Etacrynic acid 35 37 07 Renal 33 Hepatic

Na+K+2Cl-symporter inhibitors (loop diuretics)

5 Bendroflumethiazide 90 19 10 Renal 70 Hepatic

6 Chlorthiazide 685 -19 01 Renal 0

7 Chlorthalidone 94 08 1 Renal bile 25 Unknown

8 Cyclopenthiazide 913 207 9 Renal 0

9 Epithiazide 88 NA 1 Renal 15 Renal

10 Benzthiazide 460 NA 1 Renal 0

11 Hydrochlorthiazide 95 113

-01 1 Renal 0

12 Hydroflumethiazide 89 04 1 Renal 2060 Hepatic

13 Indapamide 88 27 20 Renal 100 Hepatic

14 Metolazone -16 954

18 10 Renal bile 10 Hepatic

15 Polythiazide -31 931

19 25 Renal 2575 Unknown

Na+Cl-symporter inhibitors (thiazide and thiazide-like diuretics)

16 Amiloride 87 03 1 Renal 0

17 Triametrene 62 10 01 Renal 100 Hepatic

Renal epithelial Na+ channel inhibitors (potassium-sparing diuretics)

18 Spironolactone -49 1801

28 NA Renal 100 Hepatic

19 Canrenone NA 35 NA Renal 100 Hepatic

20 Eplerenone 1511 23 NA Renal 100 Hepatic

Mineralocorticoid receptor antagonists

21 Probenecid 353 321 NA Renal 90 Hepatic Sulfonamide-derived uricosuric

Potency is relative to diuretics within the same class

REFERENCES

[1] EKJackson Diuretics In LBrunton JLazo KParker (Eds) 11th Edition Goodman and GilmansThe Pharmacological Basis of TherapeuticsMcGraw-Hill New York (2006)

[2] GJTrout RKazlauskas ChemSocRev 331(2004)

[3] RVentura JSegura JChromatogrBBiomedAppl 687 127 (1996)

[4] The List World Anti-Doping Agency (WADA) The2012 Prohibited List International Standard URLh t t p w w w w a d a -a ma o rg D o c u me n t s

World_Anti- Doping_ProgramWADP-Prohibited-l i s t To_be_effec t ive WADA_Proh ib i t ed_List_2013_ENpdf(Accessed on January 1 2013)(2013)

[5] SFCooper RMasse R Dugal JChromatogr489 65 (1989)

[6] HJGuchelaar LChandi OSchouten WAVanDen Brand Fresenius JAnalChem 363 700(1999)

[7] SJPark HSPyo YJKim MSKim J ParkJAnalToxicol 14 84 (1990)

[8] AMLisi RKazlauskas GJTrout JChromatogr581 57 (1992)

[9] ABeotra SJain TKaur Diuretics testing in sports

by GC-MSD Excretion studies ofbendroflumethiazide diclofenamide and etacrynicacid Paper presented at the International Congress

on frontiers in pharmacology and therapeutics in21st century New Delhi 3-5 Dec (1999)

[10] LAmendola CColamonici MMazzarino FBotreAnalChimActa 475 125 (2003)

[11] VSanz-Nebot IToro RBerges RVenturaJSegura JBarbosa JMass Spectrom 36 652(2001)

[12] KDeventer FTDelbeke KRoels PVan EenooBiomedChromatogr 16 529 (2002)

[13] DThieme JGrosse RLang RKMuellerAWahl JChromatogrB 757 49 (2001)

[14] MIReddy ABeotra SJain SAhi 41(2) 80(2009) WADA International Standards for Labo-ratories (ISL) Version 7 available online at httpwwwwada-amaorgdocumentsworld_anti-doping_programwadp-is aboratoriesislwada_int_standard_laboratories_2012_enpdf(Accessed on January 1 2013)

[15] TMAnnesley ClinChem 49 1041 (2003)[16] World Anti Doping Agency Technical document on

Minimum required performance limitshttpwwwwada-amaorgDocumentsWorld_Anti-Doping_ProgramWADP-IS-LaboratoriesTechnical_DocumentsWADA-TD2013MRPL-Minimum-Required-Performance-Levels-v1-2012-ENpdf(Accessed on January 1 2013)

[17] GJMurray JPDanaceau JChromatogrB 8773857 (2009)

[18] MThevis WSchanzer HSchmicklerJAmSocMassSpectrom 14 658 (2003)

[19] MBBarroso HDMeiring ADe JongRMAlonso RMJimeacutenez JChromatogrB 690105 (1997)

[20] SJPark HSPyo YJKim MSKim JParkJAnalToxico 14 84 (1990)

[21] FYTsai LFLui BChang JPharmaBiol Anal9 1069 (1991)

[22] LAmendola CColamonici MMazzarino FBotregrave

AnalChimActa 475 125 (2003)[23] JBeyer ABierl FPeters T Maurer

TherDrugMonit 27 509 (2005)[24] TGoto EMikami TOhno HJMatsumoto Food

HygieSociJapan 43(2) 95 (2002)[25] MLRiekkola JHJumppanen J Chromatogr A

31(735) 151 (1996)[26] MLu PTong HXiao SXia XZheng WLiu

Zhang L GChen Electrophoresis 28(9) 1461(2007)

[27] World Anti Doping Agency International Standardfor therapeutic use exemption Available online atVersion 5 httpwwwwada-amaorgDocumentsWorld_Anti-Doping_ProgramWADP-IS

[28] TUE2011WADA_ISTUE_2011_revJanury-2012_EN(Accessed on January 1 2013)

[29] SGuddat ESolymos AOrlovius AThomasGSigmund HGeyerMThevis WSchaumlnzer Drug

TestAnal 3 836 (2011)[30] KDeventer OJPozo PVEenoo FTDelbeke

JChromatogrA 1216(31) 5819 (2009)

270-275

277-278

279-283

Although the main application of diuretics is to en-hance renal excretion of salt and water their effects arenot limited to sodium and chloride they may also influ-ence the renal absorption and excretion of other cat-ions (K+ H+ Ca

2+ andMg

2+) anions (Cl- HCO

3- and

4-) and uric acid This pharmacological class of

drugs includes compounds with a variety of pharmaco-logical and physicochemical properties Because of thevariety of diuretic compounds classification of thesedrugs can be based on different criteria The most com-mon classification categories are by site of action in thenephron relative efficacy chemical structure effects onpotassium excretion similarity to other diuretics andmechanism of action[1]

There are several classes of diuretic drugs basedon their mechanism of actionThiazides (eg

benzthiazide) loop diuretics (eg bumetanide) potas-sium sparing diuretics (eg amiloride) carbonic anhy-

drase inhibitors (eg acetazolamide) osmotic diuret-ics (eg mannitol)and mercurial diuretics (eg mersa-lyl) (Figure 1)

Diuretics are relatively polar hence are amenableto analysis by high performance liquid chromatography(HPLC) using C18 type phases with diode array (DAD)or fluorescence detection[56] However whilst thismethod may be suitable for screening purposes it suf-fers from significant interferences due to the backgroundoccurring in urine samples The information providedby HPLC is insufficient for confirmation as alternativemass spectral confirmation of identity is needed fordoping control To overcome this problem detectionof diuretics by gas chromatography mass spectrometry(GC-MS) was introduced In order to improve thevolatility of the diuretics either methylation or silylationwas performed prior to GCMS The methylation of

these polar drugs has been the most suitable proce-

Figure 1 Site and mechanism of action of diuretics (A) The nephron with major divisions labeled (B) Mechanism ofcarbonic anhydrase inhibitors in the proximal tubule (C) Mechanism of the Na+K+2Cl- symporter inhibitors in the thickascending limb of the loop of Henle (D) Mechanism of the Na+Cl- symporter inhibitors in the distal tubule (E) Mechanismof renal epithelial Na+ channel inhibitors and mineralocorticoid receptor antagonists in the collecting duct Aldo aldosteroneCA carbonic anhydrase MR mineralocorticoid receptor Figure modified from Jackson (2006)

Full Paper

ACAIJ 13(7) 2013

EXPERIMENTAL

Sample preparation

Instrumentation

Negative polarity

Positive polarity

Recovery

Specificity

Precision

RESULTS

Full Paper

ACAIJ 13(7) 2013

Compound Ionization

mode Molecular

Product Ion (mz)

Collision Energy

1 Acetazolamide - 22225 221 83 142 -40 -35 -40 -40

2 Amiloride + 2296 230 171 116 36 43 39 39

4 Benzthiazide - 43194 430 228 308 -38 -35 -48 -48

5 Bumetanide - 36442 363 319 80 -20 -40 -40 -40

6 Canrenone - 34046 341 187 107 47 51 48 48

7 Chlorthiazide - 2957 294 214 179 -30 -42 -40 -40

8 Chlorthalidone - 3387 337 190 146 -41 -40 -35 -35

10 Etacrynic acid - 30313 301 243 207 -38 -43 -40 -40

11 Epithiazide - 425 424 268 404 -39 -18 -41 -41

12 Eplerenone - 41450 415 163 337 48 39 51 51

13 Furosemide - 3307 329 285 205 -30 -35 -40 -40

16 Indapamide - 365 364 189 132 -40 -45 -40 -40

17 Metolazone - 3658 366 259 277 34 48 47 47

18 Polythiazide - 439 438 324 418 -38 -32 -43 -43

19 Spironolactone + 4165 314 187 107 47 51 48 48

20 Probenecid + 2853 286 244 185 25 38 33 33

21 Triamterene + 2532 254 237 195 25 38 36 36

22 ACB - 28573 284 207 169 -25 -38 -30 -30

ACAIJ 13(7) 2013

DISCUSSION

Full Paper

ACAIJ 13(7) 2013

Compound LOD

(ngml) Recovery

() RSD ()

RRT- precision

(n=5X3)

Intra-day precision

2 Amiloride 20 887 71 021 45 41

4 Benzthiazide 10 1018 124 08 12 2

5 Bumetanide 10 1084 132 023 22 21

6 Canrenone 10 945 78 024 48 41

7 Chlorthiazide 20 925 89 015 59 49

8 Chlorthalidone 20 874 98 022 55 44

10 Etacrynic acid 20 874 74 015 28 25

11 Epithiazide 20 956 94 013 33 31

12 Eplerenone 10 993 101 023 34 28

13 Furosemide 10 1052 12 012 48 4

16 Indapamide 10 1045 85 009 52 5

17 Metolazone 10 110 94 013 44 5

18 Polythiazide 20 947 125 014 25 38

19 Spironolactone 10 987 13 017 35 32

20 Probenecid 10 947 95 026 44 43

21 Triametrene 10 895 89 017 55 52

22 ACB 15 781 77 01 48 44

23 ATFB 15 812 42 012 39 41

Full Paper

ACAIJ 13(7) 2013

CONCLUSION

ACKNOWLEDGMENT

Full Paper

ACAIJ 13(7) 2013

Elimination route

Metabolism

Category

2 Bumetanide 36 77

3 Furosemide 38 75

-01 1 Renal 0

REFERENCES

JChromatogrA 1216(31) 5819 (2009)

270-275

277-278

279-283

Full Paper

ACAIJ 13(7) 2013

EXPERIMENTAL

Sample preparation

Instrumentation

Negative polarity

Positive polarity

Recovery

Specificity

Precision

RESULTS

Full Paper

ACAIJ 13(7) 2013

Compound Ionization

mode Molecular

Product Ion (mz)

Collision Energy

1 Acetazolamide - 22225 221 83 142 -40 -35 -40 -40

2 Amiloride + 2296 230 171 116 36 43 39 39

4 Benzthiazide - 43194 430 228 308 -38 -35 -48 -48

5 Bumetanide - 36442 363 319 80 -20 -40 -40 -40

6 Canrenone - 34046 341 187 107 47 51 48 48

7 Chlorthiazide - 2957 294 214 179 -30 -42 -40 -40

8 Chlorthalidone - 3387 337 190 146 -41 -40 -35 -35

10 Etacrynic acid - 30313 301 243 207 -38 -43 -40 -40

11 Epithiazide - 425 424 268 404 -39 -18 -41 -41

12 Eplerenone - 41450 415 163 337 48 39 51 51

13 Furosemide - 3307 329 285 205 -30 -35 -40 -40

16 Indapamide - 365 364 189 132 -40 -45 -40 -40

17 Metolazone - 3658 366 259 277 34 48 47 47

18 Polythiazide - 439 438 324 418 -38 -32 -43 -43

19 Spironolactone + 4165 314 187 107 47 51 48 48

20 Probenecid + 2853 286 244 185 25 38 33 33

21 Triamterene + 2532 254 237 195 25 38 36 36

22 ACB - 28573 284 207 169 -25 -38 -30 -30

ACAIJ 13(7) 2013

DISCUSSION

Full Paper

ACAIJ 13(7) 2013

Compound LOD

(ngml) Recovery

() RSD ()

RRT- precision

(n=5X3)

Intra-day precision

2 Amiloride 20 887 71 021 45 41

4 Benzthiazide 10 1018 124 08 12 2

5 Bumetanide 10 1084 132 023 22 21

6 Canrenone 10 945 78 024 48 41

7 Chlorthiazide 20 925 89 015 59 49

8 Chlorthalidone 20 874 98 022 55 44

10 Etacrynic acid 20 874 74 015 28 25

11 Epithiazide 20 956 94 013 33 31

12 Eplerenone 10 993 101 023 34 28

13 Furosemide 10 1052 12 012 48 4

16 Indapamide 10 1045 85 009 52 5

17 Metolazone 10 110 94 013 44 5

18 Polythiazide 20 947 125 014 25 38

19 Spironolactone 10 987 13 017 35 32

20 Probenecid 10 947 95 026 44 43

21 Triametrene 10 895 89 017 55 52

22 ACB 15 781 77 01 48 44

23 ATFB 15 812 42 012 39 41

Full Paper

ACAIJ 13(7) 2013

CONCLUSION

ACKNOWLEDGMENT

Full Paper

ACAIJ 13(7) 2013

Elimination route

Metabolism

Category

2 Bumetanide 36 77

3 Furosemide 38 75

-01 1 Renal 0

REFERENCES

JChromatogrA 1216(31) 5819 (2009)

270-275

277-278

279-283

Recovery

Specificity

Precision

RESULTS

Full Paper

ACAIJ 13(7) 2013

Compound Ionization

mode Molecular

Product Ion (mz)

Collision Energy

1 Acetazolamide - 22225 221 83 142 -40 -35 -40 -40

2 Amiloride + 2296 230 171 116 36 43 39 39

4 Benzthiazide - 43194 430 228 308 -38 -35 -48 -48

5 Bumetanide - 36442 363 319 80 -20 -40 -40 -40

6 Canrenone - 34046 341 187 107 47 51 48 48

7 Chlorthiazide - 2957 294 214 179 -30 -42 -40 -40

8 Chlorthalidone - 3387 337 190 146 -41 -40 -35 -35

10 Etacrynic acid - 30313 301 243 207 -38 -43 -40 -40

11 Epithiazide - 425 424 268 404 -39 -18 -41 -41

12 Eplerenone - 41450 415 163 337 48 39 51 51

13 Furosemide - 3307 329 285 205 -30 -35 -40 -40

16 Indapamide - 365 364 189 132 -40 -45 -40 -40

17 Metolazone - 3658 366 259 277 34 48 47 47

18 Polythiazide - 439 438 324 418 -38 -32 -43 -43

19 Spironolactone + 4165 314 187 107 47 51 48 48

20 Probenecid + 2853 286 244 185 25 38 33 33

21 Triamterene + 2532 254 237 195 25 38 36 36

22 ACB - 28573 284 207 169 -25 -38 -30 -30

ACAIJ 13(7) 2013

DISCUSSION

Full Paper

ACAIJ 13(7) 2013

Compound LOD

(ngml) Recovery

() RSD ()

RRT- precision

(n=5X3)

Intra-day precision

2 Amiloride 20 887 71 021 45 41

4 Benzthiazide 10 1018 124 08 12 2

5 Bumetanide 10 1084 132 023 22 21

6 Canrenone 10 945 78 024 48 41

7 Chlorthiazide 20 925 89 015 59 49

8 Chlorthalidone 20 874 98 022 55 44

10 Etacrynic acid 20 874 74 015 28 25

11 Epithiazide 20 956 94 013 33 31

12 Eplerenone 10 993 101 023 34 28

13 Furosemide 10 1052 12 012 48 4

16 Indapamide 10 1045 85 009 52 5

17 Metolazone 10 110 94 013 44 5

18 Polythiazide 20 947 125 014 25 38

19 Spironolactone 10 987 13 017 35 32

20 Probenecid 10 947 95 026 44 43

21 Triametrene 10 895 89 017 55 52

22 ACB 15 781 77 01 48 44

23 ATFB 15 812 42 012 39 41

Full Paper

ACAIJ 13(7) 2013

CONCLUSION

ACKNOWLEDGMENT

Full Paper

ACAIJ 13(7) 2013

Elimination route

Metabolism

Category

2 Bumetanide 36 77

3 Furosemide 38 75

-01 1 Renal 0

REFERENCES

JChromatogrA 1216(31) 5819 (2009)

270-275

277-278

279-283

Full Paper

ACAIJ 13(7) 2013

Compound Ionization

mode Molecular

Product Ion (mz)

Collision Energy

1 Acetazolamide - 22225 221 83 142 -40 -35 -40 -40

2 Amiloride + 2296 230 171 116 36 43 39 39

4 Benzthiazide - 43194 430 228 308 -38 -35 -48 -48

5 Bumetanide - 36442 363 319 80 -20 -40 -40 -40

6 Canrenone - 34046 341 187 107 47 51 48 48

7 Chlorthiazide - 2957 294 214 179 -30 -42 -40 -40

8 Chlorthalidone - 3387 337 190 146 -41 -40 -35 -35

10 Etacrynic acid - 30313 301 243 207 -38 -43 -40 -40

11 Epithiazide - 425 424 268 404 -39 -18 -41 -41

12 Eplerenone - 41450 415 163 337 48 39 51 51

13 Furosemide - 3307 329 285 205 -30 -35 -40 -40

16 Indapamide - 365 364 189 132 -40 -45 -40 -40

17 Metolazone - 3658 366 259 277 34 48 47 47

18 Polythiazide - 439 438 324 418 -38 -32 -43 -43

19 Spironolactone + 4165 314 187 107 47 51 48 48

20 Probenecid + 2853 286 244 185 25 38 33 33

21 Triamterene + 2532 254 237 195 25 38 36 36

22 ACB - 28573 284 207 169 -25 -38 -30 -30

ACAIJ 13(7) 2013

DISCUSSION

Full Paper

ACAIJ 13(7) 2013

Compound LOD

(ngml) Recovery

() RSD ()

RRT- precision

(n=5X3)

Intra-day precision

2 Amiloride 20 887 71 021 45 41

4 Benzthiazide 10 1018 124 08 12 2

5 Bumetanide 10 1084 132 023 22 21

6 Canrenone 10 945 78 024 48 41

7 Chlorthiazide 20 925 89 015 59 49

8 Chlorthalidone 20 874 98 022 55 44

10 Etacrynic acid 20 874 74 015 28 25

11 Epithiazide 20 956 94 013 33 31

12 Eplerenone 10 993 101 023 34 28

13 Furosemide 10 1052 12 012 48 4

16 Indapamide 10 1045 85 009 52 5

17 Metolazone 10 110 94 013 44 5

18 Polythiazide 20 947 125 014 25 38

19 Spironolactone 10 987 13 017 35 32

20 Probenecid 10 947 95 026 44 43

21 Triametrene 10 895 89 017 55 52

22 ACB 15 781 77 01 48 44

23 ATFB 15 812 42 012 39 41

Full Paper

ACAIJ 13(7) 2013

CONCLUSION

ACKNOWLEDGMENT

Full Paper

ACAIJ 13(7) 2013

Elimination route

Metabolism

Category

2 Bumetanide 36 77

3 Furosemide 38 75

-01 1 Renal 0

REFERENCES

JChromatogrA 1216(31) 5819 (2009)

270-275

277-278

279-283

Compound Ionization

mode Molecular

Product Ion (mz)

Collision Energy

1 Acetazolamide - 22225 221 83 142 -40 -35 -40 -40

2 Amiloride + 2296 230 171 116 36 43 39 39

4 Benzthiazide - 43194 430 228 308 -38 -35 -48 -48

5 Bumetanide - 36442 363 319 80 -20 -40 -40 -40

6 Canrenone - 34046 341 187 107 47 51 48 48

7 Chlorthiazide - 2957 294 214 179 -30 -42 -40 -40

8 Chlorthalidone - 3387 337 190 146 -41 -40 -35 -35

10 Etacrynic acid - 30313 301 243 207 -38 -43 -40 -40

11 Epithiazide - 425 424 268 404 -39 -18 -41 -41

12 Eplerenone - 41450 415 163 337 48 39 51 51

13 Furosemide - 3307 329 285 205 -30 -35 -40 -40

16 Indapamide - 365 364 189 132 -40 -45 -40 -40

17 Metolazone - 3658 366 259 277 34 48 47 47

18 Polythiazide - 439 438 324 418 -38 -32 -43 -43

19 Spironolactone + 4165 314 187 107 47 51 48 48

20 Probenecid + 2853 286 244 185 25 38 33 33

21 Triamterene + 2532 254 237 195 25 38 36 36

22 ACB - 28573 284 207 169 -25 -38 -30 -30

ACAIJ 13(7) 2013

DISCUSSION

Full Paper

ACAIJ 13(7) 2013

Compound LOD

(ngml) Recovery

() RSD ()

RRT- precision

(n=5X3)

Intra-day precision

2 Amiloride 20 887 71 021 45 41

4 Benzthiazide 10 1018 124 08 12 2

5 Bumetanide 10 1084 132 023 22 21

6 Canrenone 10 945 78 024 48 41

7 Chlorthiazide 20 925 89 015 59 49

8 Chlorthalidone 20 874 98 022 55 44

10 Etacrynic acid 20 874 74 015 28 25

11 Epithiazide 20 956 94 013 33 31

12 Eplerenone 10 993 101 023 34 28

13 Furosemide 10 1052 12 012 48 4

16 Indapamide 10 1045 85 009 52 5

17 Metolazone 10 110 94 013 44 5

18 Polythiazide 20 947 125 014 25 38

19 Spironolactone 10 987 13 017 35 32

20 Probenecid 10 947 95 026 44 43

21 Triametrene 10 895 89 017 55 52

22 ACB 15 781 77 01 48 44

23 ATFB 15 812 42 012 39 41

Full Paper

ACAIJ 13(7) 2013

CONCLUSION

ACKNOWLEDGMENT

Full Paper

ACAIJ 13(7) 2013

Elimination route

Metabolism

Category

2 Bumetanide 36 77

3 Furosemide 38 75

-01 1 Renal 0

REFERENCES

JChromatogrA 1216(31) 5819 (2009)

270-275

277-278

279-283

ACAIJ 13(7) 2013

DISCUSSION

Full Paper

ACAIJ 13(7) 2013

Compound LOD

(ngml) Recovery

() RSD ()

RRT- precision

(n=5X3)

Intra-day precision

2 Amiloride 20 887 71 021 45 41

4 Benzthiazide 10 1018 124 08 12 2

5 Bumetanide 10 1084 132 023 22 21

6 Canrenone 10 945 78 024 48 41

7 Chlorthiazide 20 925 89 015 59 49

8 Chlorthalidone 20 874 98 022 55 44

10 Etacrynic acid 20 874 74 015 28 25

11 Epithiazide 20 956 94 013 33 31

12 Eplerenone 10 993 101 023 34 28

13 Furosemide 10 1052 12 012 48 4

16 Indapamide 10 1045 85 009 52 5

17 Metolazone 10 110 94 013 44 5

18 Polythiazide 20 947 125 014 25 38

19 Spironolactone 10 987 13 017 35 32

20 Probenecid 10 947 95 026 44 43

21 Triametrene 10 895 89 017 55 52

22 ACB 15 781 77 01 48 44

23 ATFB 15 812 42 012 39 41

Full Paper

ACAIJ 13(7) 2013

CONCLUSION

ACKNOWLEDGMENT

Full Paper

ACAIJ 13(7) 2013

Elimination route

Metabolism

Category

2 Bumetanide 36 77

3 Furosemide 38 75

-01 1 Renal 0

REFERENCES

JChromatogrA 1216(31) 5819 (2009)

270-275

277-278

279-283

DISCUSSION

Full Paper

ACAIJ 13(7) 2013

Compound LOD

(ngml) Recovery

() RSD ()

RRT- precision

(n=5X3)

Intra-day precision

2 Amiloride 20 887 71 021 45 41

4 Benzthiazide 10 1018 124 08 12 2

5 Bumetanide 10 1084 132 023 22 21

6 Canrenone 10 945 78 024 48 41

7 Chlorthiazide 20 925 89 015 59 49

8 Chlorthalidone 20 874 98 022 55 44

10 Etacrynic acid 20 874 74 015 28 25

11 Epithiazide 20 956 94 013 33 31

12 Eplerenone 10 993 101 023 34 28

13 Furosemide 10 1052 12 012 48 4

16 Indapamide 10 1045 85 009 52 5

17 Metolazone 10 110 94 013 44 5

18 Polythiazide 20 947 125 014 25 38

19 Spironolactone 10 987 13 017 35 32

20 Probenecid 10 947 95 026 44 43

21 Triametrene 10 895 89 017 55 52

22 ACB 15 781 77 01 48 44

23 ATFB 15 812 42 012 39 41

Full Paper

ACAIJ 13(7) 2013

CONCLUSION

ACKNOWLEDGMENT

Full Paper

ACAIJ 13(7) 2013

Elimination route

Metabolism

Category

2 Bumetanide 36 77

3 Furosemide 38 75

-01 1 Renal 0

REFERENCES

JChromatogrA 1216(31) 5819 (2009)

270-275

277-278

279-283

Full Paper

ACAIJ 13(7) 2013

Compound LOD

(ngml) Recovery

() RSD ()

RRT- precision

(n=5X3)

Intra-day precision

2 Amiloride 20 887 71 021 45 41

4 Benzthiazide 10 1018 124 08 12 2

5 Bumetanide 10 1084 132 023 22 21

6 Canrenone 10 945 78 024 48 41

7 Chlorthiazide 20 925 89 015 59 49

8 Chlorthalidone 20 874 98 022 55 44

10 Etacrynic acid 20 874 74 015 28 25

11 Epithiazide 20 956 94 013 33 31

12 Eplerenone 10 993 101 023 34 28

13 Furosemide 10 1052 12 012 48 4

16 Indapamide 10 1045 85 009 52 5

17 Metolazone 10 110 94 013 44 5

18 Polythiazide 20 947 125 014 25 38

19 Spironolactone 10 987 13 017 35 32

20 Probenecid 10 947 95 026 44 43

21 Triametrene 10 895 89 017 55 52

22 ACB 15 781 77 01 48 44

23 ATFB 15 812 42 012 39 41

Full Paper

ACAIJ 13(7) 2013

CONCLUSION

ACKNOWLEDGMENT

Full Paper

ACAIJ 13(7) 2013

Elimination route

Metabolism

Category

2 Bumetanide 36 77

3 Furosemide 38 75

-01 1 Renal 0

REFERENCES

JChromatogrA 1216(31) 5819 (2009)

270-275

277-278

279-283

Full Paper

ACAIJ 13(7) 2013

CONCLUSION

ACKNOWLEDGMENT

Full Paper

ACAIJ 13(7) 2013

Elimination route

Metabolism

Category

2 Bumetanide 36 77

3 Furosemide 38 75

-01 1 Renal 0

REFERENCES

JChromatogrA 1216(31) 5819 (2009)

270-275

277-278

279-283

Full Paper

ACAIJ 13(7) 2013

CONCLUSION

ACKNOWLEDGMENT

Full Paper

ACAIJ 13(7) 2013

Elimination route

Metabolism

Category

2 Bumetanide 36 77

3 Furosemide 38 75

-01 1 Renal 0

REFERENCES

JChromatogrA 1216(31) 5819 (2009)

270-275

277-278

279-283

CONCLUSION

ACKNOWLEDGMENT

Full Paper

ACAIJ 13(7) 2013

Elimination route

Metabolism

Category

2 Bumetanide 36 77

3 Furosemide 38 75

-01 1 Renal 0

REFERENCES

JChromatogrA 1216(31) 5819 (2009)

270-275

277-278

279-283

Full Paper

ACAIJ 13(7) 2013

Elimination route

Metabolism

Category

2 Bumetanide 36 77

3 Furosemide 38 75

-01 1 Renal 0

REFERENCES

JChromatogrA 1216(31) 5819 (2009)

270-275

277-278

279-283

JChromatogrA 1216(31) 5819 (2009)

270-275

277-278

279-283