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196Current Trends in Biotechnology and PharmacyVol. 6 (2) 196-203 April 2012, ISSN 0973-8916 (Print), 2230-7303 (Online)

AbstractA simple, rapid and sensitive method

using an isocratic Liquid chromatography coupledwith Tandem mass spectrometry was developedand validated for the assay of nitrofurantoin inthe Human Plasma. The Mass transition ofnitrofurantoin and losartan (Internal standard)were M/z 237.000/151.800 and M/Z 421.300/179.274 in ESI Negative ionization. Linearity wasobserved between the nitrofurantoinconcentration and the peak area ratio from10.107 to 999.900ng/mL with r2 value of 0.99.Plasma samples containing nitrofurantoin wereextracted with ethyl acetate. The observedrecovery of nitrofurantoin was 80.2 %. The intra-day and inter-day accuracy ranged from 83.61to 107.16% and from 93.13to 103.02%respectively, at Low, middle and high levelconcentrations. The method will be used in thedetermination of the pharmacokinetic parametersof nitrofurantoin after oral administration ofnitrofurantoin formulation in human Plasma.

Keywords: Nitrofurantoin, LCMS/MS,Bioanalytical, Validation, Human Plasma, 5-nitrofurans

IntroductionNitrofurantoin is an antibiotic used for the

treatment and prophylaxis for urinary tract

infections. Nitrofurantoin is more soluble in urinedue to the presence of urea and creatinine.Considerable amount of nitrofurantoin and itsmetabolites are excreted in urine. This may bethe reason; possibly, nitrofurantoin iscontraindicated in patients with a creatineclearance of 60mL/min or less, though it is notcontraindicated in rest of the population. (6, 21)Therefore, regular monitoring is required inpatients receiving nitrofurantoin. Severalmethods are reported for routine drug monitoringby using different analytical methods such as LC-MS/MS and other conventional methods (1-20).The authors suggest that performing analysis byLC-MS/MS (3) includes derivatisation techniquesand is no longer beneficial with the recentadvances in the techniques available. There is aneed for an established method for analysiswhich is not tedious and cost consuming, asderivatisation requires more reaction time as wellas more amounts of solvents and chemicalconsumption. More over it is also required to seethat the new methods being developed does notrequire more retention time so that analysis issimple and faster. Therefore, authors proposeda simple, sensitive, accurate and precise methodfor determination of nitrofurantoin by LC-MS/MSmethod for measuring nitrofurantoin plasmaconcentration.

Simple, Rapid and Sensitive Method for Determination ofNitrofurantoin in Human Plasma by using LiquidChromatography / Tandem Mass Spectrometry

Rajaram S. Patil1*, Chaitanya Krishna, A2., P. V.D.L.S.Ravi Prakash2 andSangita R. Patil2.

1Thermofisher Scientific India Pvt. Ltd. Mumbai, India2Quest life sciences Private Ltd, Chennai, India* For Correspondence - [email protected]

Nitrofurantoin in Human Plasma by using Liquid Chromatography / Tandem Mass Spectrometry


Stricture of Nitrofurantoin

Materials and MethodsMaterials: Nitrofurantoin and Losartan

analytical standards were obtained from BioconIndia Pvt Ltd, HPLC grade Acetonitrile andmethanol were obtained J.T. Baker, USA,Ammonium acetate (GR Grade Merck), Dimethylsulphoxide (AR Grade Merck), Orthophosphoricacid (GR Grade Merck),Glacial acetic acid(GRGrade Merck ),Water was de-ionized and tripledistilled.

Preparation of Nitrofurantoin standardsolutions: Standard solutions of Nitrofurantoin

(1.0 mg/ml) was prepared by dissolving anaccurately weighed amount of 10.00 mg ofNitrofurantoin in 5 ml of Acetonitrile and add 50µL of Dimethyl sulphoxide, sonicated for 5minutes and the volume was adjusted to 10 mlwith Acetonitrile. The standard solution wasstored subsequently at 2-8 0C. The appropriateconcentrations of standard solution wereprepared by diluting the stock solution with 80:20 Acetonitrile: water.

Preparation of Losartan Internal standardsolutions: Standard solution of Losartan (1.0 mg/ml) was prepared by dissolving an accuratelyweighed amount of 10.00 mg of Losartan in 5 mlof methanol, sonicated for 5 minutes and thevolume was adjusted to 10 ml with methanol. Thestandard solution was stored subsequently at 2-8 0C. 10.00µg/mL Losartan was prepared bydiluting the stock solution with 80: 20 methanol:water.

Sample preparation

Thaw (30 min) Plasma Sample

Add 2.5 mL Ethyl

Acetate

Centrifuge at 4500 rpm for

10 minutes at 5°C

Add 50 µL of ISTD

Evaporate the supernatant at 400C With Nitrogen Collect 1.8 mL of supernatant

Reconstitute the residue with 5mM Ammonium Acetate: Acetonitrile (50:50%)

(50:50%) and inject 10ul into LC-MS/MS.

Vortex for 5 min

Acidfy with 50µL of 10 %

Orthophosphoric acid

Vortex (1 min)

Vortex (1 min)

Instrumentation and conditions : The ThermoScientific HPLC (Model: Surveyor) and LC-MS/MS system interfaced to LC Quan Software in awindows platform The Reverse phase C8analytical column (Kromosil C 8, Length: 4.6×50mm, Particle size: 5 µ) was used for theseparation of Nitrofurantoin and internal standard,the mobile phase consists a mixture of

Acetonitrile and 5mM Ammonium Acetate at aratio of 60:40 respectively. The mobile phase wasdegassed by passing through a 0.22 µmmembrane filter (Millipore, Bedford, MA, USA)prior to use and operated through a single pump(Isocratic) at a flow rate of 0.5 ml/min. The injectorwas filled with an injector loop of 10 µl. Tandemmass spectrometric detection and quantification

Rajaram S. Patil et al


was performed using multiple reaction monitoring(MRM).

Results and DiscussionDevelopment conditions for rapid extractionof Nitrofurantoin from Human Plasma:Reported extraction techniques for determinationof nitrofurantoin from human plasma [3-6, 8-11,14-19] is more laborious and involves moretedious process like derivatisation. More over,they involve cost efforts and in addition to this,they are time consuming. While developing amethod, one needs to look at thechromatographic conditions and extractionprocess for minimizing the disadvantages of oldertechniques or methods. In the process, mobilephase selection and optimization, columnselection is critical to minimize run time, solventconsumption and injection volumes. It wasobserved that, a novel method can be developedwith advantages that will eliminate derivatisation,more solvent consumption, high run time andrising costs. The new method should offer thebenefits of using small columns, reducing plasmaand solvent consumption to arrive at an extractionprocedure which will have more extractionrecoveries. Conditions for simple and rapid HPLCseparation with MS/MS Electro-spray Negativeionization mode were developed using anisocratic elution with a mobile phase composedof Acetonitrile and 5mM Ammonium acetate at aratio of 60:40% v/v. Thus the ions formed for drugand Internal standard in ESI Negative mode dueto the addition of Hydroxyl ion (OH-) in carbonylfunction (C=O) present in the drug (Fig.1). Theseconditions gave a well defined, sharp peak ofNitrofurantoin and Losartan (ISTD) with aretention time of approximately 1.21 and1.51minutes. Under these conditions an amountof Nitrofurantoin as low as 1ng/mL could bedetected. With these retention times, analysiscould be completed in about 3.0 minutes.

Method validationLinearity: The quantification of thechromatogram was performed using the peak

area ratio of Nitrofurantoin and Losartan (ISTD).Nine standard solutions were prepared.

(10.248, 25.621, 51.242, 102.484, 256.209,457.515, 667.906, 861.814 ng/mL and 1013.899ng/mL) and subjected analyses by HPLC-MS/MS. Three precision and accuracy (P&A) batcheswere injected. The peak area ratio wasdetermined and plotted versus the concentrationof Nitrofurantoin. Statistical analysis using leastsquare regression analysis indicated excellentlinearity for Nitrofurantoin with the concentrationrange studied as shown in Table 1. In constructingthe standard curve, samples of Nitrofurantoin inHuman Plasma identical to those in the standardsolutions were prepared and the Nitrofurantoinresponse ratios were plotted against theconcentrations of Nitrofurantoin in ng/mL asshown in Fig. 2. The linearity of the concentrationand response relation was established over therange of 10.248 – 1013.899 ng/mL (R2 = 0.9898).Fig. 3 shows the LC-MS/MS chromatograms ofpure drug. (Nitrofurantoin), Fig. 4 shows the LC-MS/MS chromatograms of drug-free Humanplasma and Fig. 5 shows the LC-MS/MSchromatograms of standard Plasma samplecontaining the drug at a concentration of10.248ng/mL.

Accuracy and precision: The intra-dayaccuracy and precision of the assay wasevaluated by analyzing six replicates of thePlasma containing Nitrofurantoin at threedifferent concentrations. The intra-day precisionsof the analyzed samples are determined byRelative Standard Deviation (RSD) range from2.11% to 11.01%, while the intra-day accuracyranged from 83.61% to 107.16%. The inter-dayprecision of the assay was measured byanalyzing six replicates of Nitrofurantoin Plasmasamples for three consecutive days. The inter-day precision of the analyzed samples asdetermined by Relative Standard Deviation(RSD) range from 6.48% to 12.37%, while theinter-day accuracy ranged from 93.13to 103.02%.


;


Recovery: The absolute recovery wascalculated by comparing the peak areas ofNitrofurantoin and Losartan standards to thoseassessed by extraction of Nitrofurantoin andLosartan at the three different concentrations.Results of absolute recovery of Nitrofurantoinand Losartan were 103.160% and 94.889%respectively as shown in Table 2.

ConclusionThis method was validated in human

Plasma for its specificity, sensitivity, linearity,accuracy, precision (repeatability &reproducibility), % recovery, stability of samples(Freeze thaw, Bench top and Auto samplerstability, short-term and long term stability of stocksolution and Internal Standard), dilution integrityand for ruggedness. Method is applicable toquantify the Nitrofurantoin in clinical samples forfurther evaluation of pharmacokinetics.

AcknowledgementThe author would like to thank

Dr.S.S.Makone and Dr.Vinay P. Shedbalkar fortheir continuous encouragement and remarks.

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ble

1. C

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amet

er s

umm

ary

and

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-cal

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ted

calib

ratio

n cu

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13.8

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CC

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5733

337

26.9

9888

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.4 4

9777

7110

5.37

2637

270.

9323

7843

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7814

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6387

676

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2110

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4434

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2)

10.5

0949

324.

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5064

9.65

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105.

2333

1826

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943

5.93

4921

682.

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9761

837.

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386

1032

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0417

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421

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9938

440.

858

154

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842.

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1027

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AN

10.2

382

25.1

0064

9.50

7410

6. 8

113

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4 37

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4281

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0310

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2.57

661

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8724

44.9

3553

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3553

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QC ID LOQQC LQC MQC HQCActual (ng/ml) 10.3129 28.2546 357.6527 861.8138QC-PA1 8.03635398 27.065301 356.704618 893.1308

8.41787092 30.770766 342.647412 864.65738.35451263 36.393436 361.322031 866.00978.94058529 28.173236 357.13257 879.2019.02181804 29.208245 364.6488 833.92738.96339905 29.028796 357.699024 811.64

QC-PA2 9.7851 31.2179 322.2158 806.162810.5656 33.7762 322.0735 707.44979.8086 29.6063 325.5442 790.01079.1712 29.3972 319.8673 727.55729.4565 29.6731 314.3319 780.19888.3166 23.9637 285.4347 665.0917

QC-PA3 11.0094524 26.863036 345.577665 781.798411.4023056 25.54112 365.126322 783.584511.6242023 24.274589 362.855776 842.741710.8603541 30.151343 338.160634 806.935911.7378058 28.922102 352.064364 827.81959.6742967 29.920648 354.000862 778.6509

Mean 9.7304 29.1082 341.5226 802.5871SD 1.20344 3.04063 22.13759 59.85233%CV 12.37 10.45 6.48 7.46%Nominal 94.35 103.02 95.49 93.13


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Table 2. Within and between batch precision and accuracy

Acceptance criteria1) The within and between batch mean Precision for LQC, MQC and HQC samples should be within

15% and 20% for the LOQ QC2) The within and between batch mean Accuracy for LQC, MQC and HQC samples should be within 85-

115% and 80-120% for the LOQ QC


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