Research Article

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LC-coupled ESI MS for quantification of miltefosine in human and hamster plasma

Background: Leishmaniasis, a fatal parasitic disease, is the second largest parasitic killer in the world and miltefosine is the first and only oral drug available for its treatment. Results: A rapid, sensitive and simple LC–MS/MS method for miltefosine quantification in hamster and human plasma was developed and validated over the range of 2.5–400 ng/ml. The mass spectrometric detection of the drug was carried out in multiple reaction monitoring mode (m/z 408.1→125.1) using an electrospray positive ionization. The protein precipitation method was employed for sample (50 μl) cleanup. Conclusion: The proposed method provided accurate and precise highthroughput quantification of miltefosine in plasma and was successfully applied to its oral PK study in Golden Syrian hamsters. First draft submitted: 21 July 2015; Accepted for publication: 6 January 2015; Published online: 26 February 2016

Swati Jaiswal1,2, Abhisheak Sharma1,2,3, Mahendra Shukla1,2 & Jawahar Lal*,1,2 Pharmacokinetics & Metabolism Division, CSIR-Central Drug Research Institute, Lucknow 226031, India 2 Academy of Scientific & Innovative Research, New Delhi, India 3 Department of Pharmaceutics & Drug Delivery, The University of Mississippi, MS 38677, USA *Author for correspondence: Tel.: +91 522 2772474 Fax: +91 522 2771941 j_lal@ cdri.res.in 1

Keywords: HPLC–MS/MS • leishmaniasis • miltefosine

Leishmaniasis is a neglected tropical and subtropical disease caused by trypanosomatids from the genus Leishmania, which are transmitted to humans by the bite of infected female phlebotomine sand flies [1] . As per WHO, an estimated 1.3 million new cases of visceral and cutaneous leishmaniasis are reported annually and 20,000–30,000 deaths occur worldwide each year [2] . India witnessed the occurrence of 50% of the world’s leishmaniasis cases and 70% of those are in Bihar [3] . Poverty-allied malnutrition, poor housing and sanitary conditions, weak immune system and lack of resources are the major risk factors associated with the leishmaniasis [2] . Transmission of the leshmanial parasite occurs in 88 tropical and subtropical countries where the sand fly vector is present subjecting around 350 million people toward risk of contracting the disease [2] . Leishmania parasite lives a digenetic life cycle. It exists in promastigote and amastigote form in insect and human vector, respectively. The phlebotomine sand

10.4155/bio.16.7 © 2016 Future Science Ltd

flies are exclusively responsible for transmission as well as proliferation of promastigotes. After biting by phlebotomine sand flies, the parasite multiplies within mammalian macrophages, dendritic cells and/or neutrophils as intracellular amastigotes [4] . Miltefosine, 2-(hexadecoxy-oxido-phosphoryl) oxyethyltrimethyl-azanium, is the first reported oral drug for both visceral and cutaneous leishmaniasis  [5] . It is an alkylphosphocholine which is amphiphilic in nature and affects several lipid metabolic pathways in Leishmania parasite [6] . It causes apoptosis like death in Leishmania donovani promastigotes as well as in extra- and intra-cellular amastigotes leading to potent activity against leishmaniasis  [7,8] . Miltefosine is reported to get metabolized by phospholipases and major metabolites identified are choline, phosphocholine and diacyllecithin [9,10] . Miltefosine is registered in Germany, several countries in South America and Indian subcontinent for the treatment of visceral and cutaneous leishmaniasis and is included in the

Bioanalysis (Epub ahead of print)

part of

ISSN 1757-6180

Research Article  Jaiswal, Sharma, Shukla & Lal WHO essential medicines list as an anti-leishmaniasis ­medicine in 2011 [11] . In view of the clinical importance of miltefosine, a simple, selective, sensitive, fast and robust bioanalytical method is required. Few methods are available for miltefosine analysis in biological matrices but encompass limitations  [12,13] . An HPLC method has been reported for quantification of miltefosine using evaporative lightscattering detector [12] . The evaporative light-scattering detector-based detection methods have adequate sensitivity but are uncommon, which seem to be inept in the present scenario of advanced technology. One LC–MS/ MS-based bioanalytical method has also been reported for quantification of miltefosine in human plasma, which requires a larger volume of plasma, utilizes a more time-consuming and costlier sample preparation with solid-phase extraction (SPE) [13] . In order to overcome the shortcomings, a new bioanalytical method using LC–MS/MS for quantification of miltefosine is developed. The developed method is superior to the previously reported methods as it is more sensitive, posses short chromatographic run time, requires a lower volume of plasma (50 μl) and utilizes a more rapid, simple and inexpensive sample preparation technique (Table 1). The method will be useful for performing advanced preclinical and clinical PK–PD modeling studies. It can be advantageous during therapeutic drug monitoring (TDM) in special patient population (newborns, children and pregnant women), where withdrawal of high blood volume is not recommended and considered unethical. The short chromatographic run time (4.5 min) allows analysis of more than 150 samples per day and can provides benefit in high-throughput bioanalysis. Experimental Materials & reagents

The miltefosine and phenacetin (internal standard, IS) and LC–MS grade acetonitrile (ACN) and metha-

nol (MeOH) were purchased from Sigma-Aldrich (St. Louis, USA). Heparin injection IP (NEPORIN, 25000 IU/5 ml) was procured from the Biocon (Bangalore, India). Microcentrifuge tubes were obtained from Tarsons Product Pvt Limited, India. Ultrapure water of resistivity 18.2 MΩ cm at 25°C was from Milli-Q PLUS PF (Billerica, USA) water purification system. Human blood (drug free) was collected in heparinized tubes by clinician in-house from six healthy male volunteers (age: 25–30 years; weight: 70 ± 10 kg) after obtaining their consent for bleeding. Similarly, hamster blood (drug free) was obtained from six young and healthy male hamsters (age: 5–6 weeks; weight: 50 ± 10 gm) provided by the National Laboratory Animal Centre (NLAC) of the institute. Plasma was separated after centrifuging the collected blood at 3000× g for 10 min and stored in glass tubes at -80°C till use. Routinely, we use glass tubes for storing biosamples due to the problems of plausible leaching from plastic. All experiments, euthanasia and disposal of carcasses were carried out as per the guidelines of Institutional Animal Ethics Committee at the CSIR-Central Drug Research Institute (IAEC approval no.: IAEC/2015/14 dated 15 April 2015). HPLC equipment & conditions

A Shimadzu series UFLC system (Shimadzu, Kyoto, Japan) consisting of a prominence pump (Model LC20AD) with online degasser (Model DGU-20A3) was used to deliver the mobile phase (MeOH:Milli-Q water [90:10, %v/v] with 0.2% formic acid) at a flow rate of 0.8 ml/min. The samples were injected through an autosampler (Model SIL-HTc) with a temperaturecontrolled (4°C) peltier tray onto the mass spectrometer. Chromatographic separations were achieved on a Unisol Amide column (100 × 4.6 mm, 3 μm; Agela Technologies, USA). The column oven (CTO‐10AS) temperature was 30°C. ACN:Milli-Q water (50:50,

Table 1. Comparison of proposed assay for quantification of miltefosine with previously reported methods. Method

Dorlo et al.†

Lemke and Kayser‡

Detector

MS/MS

Evaporative light-scattering detector MS/MS

Linearity

4–2000 ng/ml

300–2500 ng/ml

2.5–400 ng/ml

LLOQ

4 ng/ml

300 ng/ml

2.5 ng/ml

Sample volume

250 μl

1 ml

50 μl

Extraction technique

Solid-phase extraction Solid-phase extraction

Protein precipitation

Recovery

>82%

>87%

>98%

Injection volume

20 μl

25 μl

10 μl

Run time

7 min

5 min

4.5 min

† ‡

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Proposed assay

[13]. [12].

Bioanalysis (Epub ahead of print)

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LC-coupled ESI MS for quantification of miltefosine in human & hamster plasma

Research Article

Table 2. Optimized multiple reaction monitoring parameters for analyte and internal standard. Analyte  

Compound parameters Q1

Q3

DP (V)

EP (V)

CE (V)

Source parameters CXP (V)

CG (psi) ISV (V)

T (°C)

GS1 (psi) GS2 (psi)

Miltefosine

408.10

125.10

110.00

10.00

40.00

10.00

10.00

4500.00 550.00 45.00

50.00

Phenacetin (IS)

180.10

138.20

60.00

10.00

25.00

10.00

 

 

 

 

 

CE: Collision energy; CG: Curtain gas; CXP: Collision cell exit potential; DP: Declustering potential; EP: Entrance potential; ISV: Ion spray voltage; GS1: Nebulizer gas; GS2: Heater gas; Q1: Parent ion; Q3: Product ion; T: Temperature.

%v/v) was used as rinsing solution and rinsing mode was set to before and after aspiration to minimize carryover, if any. LC parameters like needle rinsing speed and volume, needle stroke, sampling speed, purge time and rinse dip time were set at 35 μl s-1, 500 μl, 52 mm, 5 μl s-1, 0.5 min and 10 s, respectively. MS

Mass spectrometric detection was performed on an API 4000 Q Trap mass spectrometer (Applied Biosystems, Toronto, Canada) equipped with Turbo V ion source operated using standard ESI coupled with the LC separation system. Multiple reaction monitoring was used to perform mass spectrometric quantification of miltefosine and IS using ESI in the positive mode. The parent ion to daughter ion transitions were monitored from m/z 408.1→125.1 and 180.1→138.2 miltefosine and IS, respectively. The source parameters (curtain gas, ion spray voltage, source temperature, turbo ion spray nebulizer gas and turbo heater gas) and the compound parameters (declustering potential, collision energy, entrance potential and collision exit potential) for miltefosine and IS are optimized with a rationale of increasing the signal intensity and the finally selected parameters are listed in Table 2. Both Q1 and Q3 quadrupoles were operated at unit resolution and dwell time for both miltefosine and IS was set at 200 ms. Zero air was used as the nebulizer gas, while nitrogen was employed as both curtain and collision gas. The total analysis time was 4.5 min per sample. Analyst software (Version 1.4.2; Applied Biosystems) was used for control of the equipment, data acquisition and analysis.

and IS. The CS samples were prepared by spiking WS (≤2.5%) in drug-free hamster and human plasma followed by appropriate dilution to obtain 2.5, 5, 10, 25, 50, 100, 200, 380 and 400 ng/ml of miltefosine. The QC samples were prepared in sextuplicates at four concentration levels, viz. 360 ng/ml (high-quality control, HQC), 180 ng/ml (medium-quality control; MQC), 3.75 ng/ml (low-quality control; LQC) and 2.5 ng/ml (LLOQ). The concentrations of QC samples are selected as per the US FDA guidelines [14] . Similarly, the dilution integrity quality control (DIQC) samples containing 36 μg/ml of miltefosine were prepared by spiking 3.6 μl of SS to 96.4 μl drug-free hamster and human plasma. A 100- and 200-fold dilution was prepared by spiking 2 and 1 μl of DIQC (n = 6) in 200 μl drug-free hamster/human plasma, respectively. WS containing 1 μg/ml of the IS was prepared by appropriate dilution of its stock solution (100 μg/ml) in ACN. All the stock (SS and WS) solutions were stored at 4°C and vortex-mixed before use. Sample cleanup

A simple and single-step protein precipitation method was employed for the extraction of miltefosine from hamster/human plasma. Aliquots (50 μl) of drug-free, spiked or test plasma samples were transferred in a 1.5 ml microcentrifuge tubes and 250 μl of mobilephase containing IS (10 ng/ml) was added. The samples were vortexed for 5 min and centrifuged at 3000 rpm for 10 min at 4°C. The clear supernatant (100 μl) was separated and transferred into prelabeled autosampler vial and 10 μl was injected onto the analytical column and assayed using the LC–MS/MS system.

Stock & standard solutions preparation

The standard stock (SS, 1 mg/ml) of miltefosine was prepared by dissolving the weighed amount in ACN. Working stock (WS) solutions containing 100 μg/ml of miltefosine were prepared by appropriately diluting the SS in ACN. The calibration standards (CS) and quality control (QC) samples were prepared in drug-free hamster and human plasma by serial dilution methodology using independent WS of miltefosine. Pooled drug-free hamster and human plasma was prescreened to assure the absence of any endogenous interference at the retention times of both the analyte

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Method validation

The method was validated to demonstrate the selectivity, matrix effect, specificity, accuracy, precision, linearity, recovery, dilution integrity and stability as per the FDA bioanalytical method validation guidance [14] . Incurred sample reanalysis was also performed to increase the reliability of the developed method. Selectivity

The selectivity was examined by analyzing the blank plasma from six different hamsters/humans. Chro-

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Research Article  Jaiswal, Sharma, Shukla & Lal matogram from each of these drug-free plasma samples was examined for the presence of endogenous plasma components eluting at the retention times of miltefosine and IS. Selectivity was assured if the peak response from endogenous plasma components was less than 20% of the mean peak response of miltefosine at LLOQ level and less than 5% of the mean peak response of IS. Sensitivity

For evaluation of method sensitivity, replicates of hamster and human plasma spiked with miltefosine were processed and analyzed. The LOD was determined based on analyte’s S/N ratio of 3:1. The S/N ratio was calculated using Analyst’s script (S_NstdDevQS) with standard deviation 3. The LLOQ was defined as the lowest concentration at which analyte’s response is at least five-times the response compared with blank response. Analyte’s peak at LLOQ must be identifiable, discrete and reproducible with accuracy and precision within 20%. Calibration curve & linearity

The calibration curve includes a blank sample (drugfree plasma with IS only), a double blank sample (drug-free plasma without IS) and nine calibration standards (plasma containing 2.5, 5, 10, 25, 50, 100, 200, 380 and 400 ng/ml of miltefosine and 10 ng/ml of IS). The calibration curve was constructed by plotting the peak area ratio of miltefosine to that of IS (A Miltefosine /A IS) against the corresponding nominal concentration of calibration standards. The A Miltefosine /A IS versus nominal concentrations plot was subjected to least-square linear regression with (1/x and 1/x 2) and without weighting factor. The acceptance criteria for each back-calculated standard concentration was set at ±15% deviation except for the LLOQ, where it was set at ±20% deviation of the nominal concentration. For typical LC–MS/MS analysis, we used one calibration curve with QCs interspersed within the test samples and the acceptance criterion for the standard curve is that at least 75% of nonzero standards should meet the above criteria, including the LLOQ. Matrix effect & carryover effect

Ion suppression or enhancement of miltefosine and IS owing to matrix components associated with hamster and human plasma was assessed both qualitatively (postcolumn infusion) and quantitatively (postextraction addition) at three QC (LQC, MQC and HQC) concentrations (each in sextuplicates). Plasma from six different hamsters and human was processed using the aforementioned protein precipitation method. WS (equivalent to LQC, MQC and HQC) was spiked

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into the processed blank matrix. Furthermore, to check for lot-dependent difference in matrix effect, LQC is prepared in processed blank matrix from six unique lots of hamster plasma. Analytical standards of equivalent LQC, MQC and HQC concentrations were also prepared in mobile phase. The mean peak area of each postextracted spiked sample was compared with that of respective analytical standards at QC level. The acceptable limit for the matrix effect was set to be within the range of 85–115%. For investigating the influence of the matrix on analyte response over the entire chromatographic run, postcolumn infusion analysis was carried out. An infusion pump was used to deliver a constant flow of analyte into the HPLC eluent at a point after the chromatographic column and before the mass spectrometer ionization source. A sample of matrix from extracted drug-free human/hamster plasma (without analyte) was injected under the desired chromatographic conditions and the response from the infused analyte was recorded. The carryover effect was examined by injecting the processed double blank plasma sample just after the ULOQ sample (400 ng/ml) at the end of calibration standards run. For double blank plasma samples, the peak area of analyte and IS should be less than 20% of the response at LLOQ. Accuracy, precision & recovery

Accuracy and precision of the method were determined by replicate analysis of QC samples (n = 6, each concentration) prepared at four different concentrations (HQC, MQC, LQC and LLOQ) for five consecutive days. Although, QC runs for precision and accuracy are required for 3 days only; however, it was continued for 5 days. The accuracy (intra- and inter-assay) was calculated as %bias = ([observed concentration – nominal concentration]/nominal concentration) × 100. Intra- and inter-assay precision, in terms of percent relative standard deviation (%RSD), was calculated from %RSD = ([√MS]/mean observed concentration) × 100 by subjecting the data to one-way analysis of variance (ANOVA), where MS is the mean square value. Extraction recovery of miltefosine was categorized in a dual manner. True recovery is estimated by comparing the peak area response obtained from extracted spiked hamster or human plasma control samples (LQC, MQC and HQC, each n = 6) against respective postextraction spiked standards. Likewise, combined recovery is calculated by comparing extracted spiked plasma control samples against respective analytical standards. The recovery of IS was also determined similarly at a concentration of 10 ng/ml. True and combined recoveries were calculated from True Recovery (%) = (peak area of analyte after extraction/peak area of postextrac-

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LC-coupled ESI MS for quantification of miltefosine in human & hamster plasma

tion spiked standards) × 100 and Combined Recovery (%) = (peak area of analyte after extraction/peak area of analytical standards) × 100, respectively. Dilution integrity

The capability to dilute samples with concentrations above the ULOQ was also investigated. Plasma DIQC standards (n = 6) with 36 μg/ml miltefosine were 100and 200-times diluted with either drug-free hamster or human plasma, then processed and assayed. The mean observed concentration was compared with the nominal value. The accuracy and precision had to be within a range of 85–115%. Stability

The chemical stability of miltefosine and IS in stock solutions (SS and WS) was assessed for storage in refrigerator (at 4°C) for 45 days. The peak area responses of miltefosine and IS in analytical standards containing 3.75, 180 and 360 ng/ml of miltefosine and 10 ng/ml of IS (each n = 6) from freshly prepared stock solutions and the stored stock solutions were compared. The samples were considered stable when the difference between the two was less than 5%. The stability of the analyte in the hamster and human plasma was evaluated by using an array of stability experiments such as bench-top stability (6 h at ambient temperature), freeze–thaw stability (up to three freeze [-80°C, for 7 days] – thaw [room temperature, 30 min] cycles), long-term stability (-80°C for 30 days) and autosampler stability (10°C for 24 h). Each stability experiment was carried out using sextuplicates of QC samples at three concentrations (LQC, MQC and HQC). An acceptance criterion for each stability study was ±15% deviation from the nominal concentration drawn against the freshly prepared calibration curve. Incurred sample reanalysis

Further to show the reproducibility of method, once assayed in vivo PK study samples were selected (n = 4 from each hamster) and reassayed. The selected samples included the samples around the Cmax and from the elimination phase to cover the entire profile of the individual animal. Thus, the incurred sample reanalysis (ISR) was performed with 12 out of total 48 samples. The previously determined concentrations of these samples served as the reference. The change in concentration was determined by comparing the concentrations after reanalysis with the reference concentration and was expressed as %difference = ([reanalyzed concentration – reference]/mean of reference and reanalyzed concentration) × 100, which should be within 20% for at least two-third of the total reanalyzed samples.

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Research Article

Application to PK study of miltefosine in Golden Syrian hamsters

The validated LC–MS/MS assay method was applied to single-dose PK study of miltefosine in Golden Syrian hamsters (Mesocricetus auratus), which is considered as an appropriate experimental model to study visceral leishmaniasis because it is highly susceptible to infection with Leishmania donovani and reproduces the clinicopathological and immunopathological features remarkably similar to human disease [15] . Young and healthy male Golden Syrian hamster (n = 3) weighing 50 ± 10 g was obtained from Laboratory Animal Division of the Institute. The animals were acclimatized at least a week prior to commencement of the study and were maintained on standard food and water ad libitum. A solution formulation of the compound was prepared by dissolving the weighed quantity of miltefosine in Milli-Q water. In this study, the hamsters received a single 10 mg/kg oral dose of miltefosine. The animals were provided with standard diet 2 h after dosing. Blood samples (≈150 μl) were carefully collected under light ether anesthesia from the retro-orbital plexus into heparinized microcentrifuge tubes at 0.5, 1, 2, 4, 6, 10, 24, 48, 72, 96, 120, 168, 216, 312 and 408 h postdosing. The blood samples were centrifuged at 3000 rpm for 10 min; the plasma was separated into clean and labeled tubes and stored at -80°C until analysis. The hamster plasma samples (50 μl) were assayed along with calibration standards and QC samples and the levels of miltefosine were calculated using Analyst software. PK analysis

The plasma concentration–time data were subjected to noncompartmental approach using Phoenix WinNonlin (version 6.3; Certara Inc, MO, USA) [16] . All the experimental data and PK parameters are expressed as the mean ± standard error mean (SEM). From the observed concentration versus time data, the peak plasma concentration (Cmax) and the time to reach Cmax (Tmax) were obtained directly. The area under the plasma concentration–time curve from time zero to the time of final measurable sample (AUClast) was calculated using the linear trapezoidal method. Terminal half-life (t1/2) is calculated from t1/2 = ln(2)/λz, where λz is the first-order rate constant associated with the terminal (log-linear) portion of the curve and is estimated by linear regression of time versus log concentration. Results & discussion Method development

The method development started with optimization of mass spectrometric ionization and fragmentation of miltefosine and IS (each 100 ng/ml) separately via infusion using Gaslight syringe (Hamilton Company, NV,

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Research Article  Jaiswal, Sharma, Shukla & Lal USA) and Harvard Pump 11plus (Harvard Apparatus, Holliston, USA) into electrospray ionization source. Phenacetin was used as IS as it possesses comparable physicochemical properties with miltefosine. It is soluble in methanol, retained on amide column and elutes with good peak shape and intensity at retention time close to miltefosine, which is also good as it will cover up the variations in analyte response due to any variation in instrument. Tuning of mass parameters was performed both in positive and negative ionization mode. Better responses were observed in the positive mode for both miltefosine and phenacetin. Both showed only one prominent ion, which corresponds to the protonated [M + H]+ ion. So, for miltefosine and IS, [M + H]+ ion was selected as the parent ion (Q1) and used as precursor ion to obtain product ion (Q3). The product ion mass spectra of miltefosine and IS are presented in Figure 1. Multiple reaction monitoring technique was selected for the method development and the parameters were optimized to maximize the response for miltefosine. Two most sensitive mass transitions selected for miltefosine were m/z 408.10→125.10 and 408.10→184.12, among these former one is used for quantitative analysis. The mass transition selected for IS was m/z 180.1→138.2. In pursuit of a good peak shape and a short retention time (2–4 min), liquid chromatographic conditions were scrutinized using various types of column (amide, Cyano, C-18, C-8), mobile-phase compositions (ACN and MeOH as organic modifiers with different buffers viz ammonium acetate, ammonium formate and formic acid with variable pH range 3.0–6.8) and flow rate (0.4–1.0 ml/min). Poor retention and improper peak shape of miltefosine were obtained on C-18 and C-8 columns. Cyano column provided good retention of miltefosine; however, peak tailing was observed. With amide column, adequate retention was observed with slight peak fronting. The peak fronting was eliminated with use of 0.2% v/v formic acid in aqueous phase in combination with 90% v/v methanol. There was no improvement in the retention of miltefosine on changing the organic modifier in mobile phase. Amide column exhibited best sensitivity, efficiency and peak shape using mobile phase (MeOH:Milli-Q water [90:10, %v/v] with 0.2% formic acid) at a flow rate of 0.8 ml/min. After optimization of chromatographic conditions, a suitable extraction method was required for adequate recovery of the analyte from biological matrices. The simple and convenient protein precipitation and liquid–liquid extraction (LLE) approach utilizing various combinations of extraction solvents in different ratios were tried to obtain a maximum and consistent recovery with the minimal matrix effect. Initially, LLE was tried by using four different extraction solvents (diethyl ether, ethyl acetate, 3% isopro-

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pyl alcohol in n-hexane and 60% n-hexane in ethyl acetate). The extraction recovery of miltefosine varied between 5 and 20%. Protein precipitation of plasma samples using methanol and acetonitrile provided better recovery (80–90%) than LLE. However, maximum recovery (>98%) and better peak shape were obtained when mobile phase was used for protein precipitation. Considering the rationale of cost-effective method development, SPE was not tried. In comparison to SPE and LLE, protein precipitation is simple, rapid and cheap extraction method and also provided good recovery of miltefosine. Though protein precipitation is the dirtiest sample cleanup but with amide column, we obtained better peak shape of miltefosine and lesser matrix effect was observed for both miltefosine and IS. Method validation

Representative chromatograms of extracted blank and drug spiked plasma are shown in Figure 2. Miltefosine and IS eluted at 1.79 and 1.57 min, respectively. Selectivity of the method was guaranteed as no significant interference from endogenous plasma constituents was detected in blank hamster or human plasma at retention times of either miltefosine or IS (Figure 2) . The LOD for miltefosine was 1 ng/ml (S/N ratio ≥4.4) and LLOQ was 2.5 ng/ml (S/N ratio ≥11.2). The analyte response at LLOQ was greater than five-times the blank response. The analyte peak at LLOQ was identifiable, discrete and reproducible (Figure 2 & Table 3) . No coeluting peaks of area more than 20% of analyte area at the LLOQ level and greater than 5% area of IS were observed in the chromatograms of processed blank plasma samples from six different lots of plasma (hamster). The retention time and peak area showed minimal variability (%RSD, 500 ng/ml may cause contamination in Q0 region of LC–MS/MS system and carryover effect in subsequent runs, ULOQ was selected as 400 ng/ml. The calibration curves with intercepts applying a weighted 1/x2 linear regression analysis were reliable and reproducible with lowest percentage relative error during 5 days’ validation period. All the calibration standards were within the acceptance criteria (accuracy of ±20% for LLOQ and ±15% for non-LLOQ standards) and the regression coefficient values were always ≥0.996. A typical calibration curve for miltefosine has the regression equation of y = (0.00495)x + (0.0217), r2

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LC-coupled ESI MS for quantification of miltefosine in human & hamster plasma

125.10

2.6e5

H+

OH O

2.4e5

P

Research Article

O

O-

2.2e5 Product ion m/z, 125.10

2.0e5

184.12

1.8e5 Intensity (cps)

O

1.6e5

H+

OH

-

N+

H3C

1.2e5

O

O

H3C

1.4e5

P

CH3

Product ion m/z, 184.12

H+

1.0e5 O

8.0e4

H 3C H3C

4.0e4

P

O

O-

N+

6.0e4

CH3

(Parent ion m/z, 408.10)

85.91

2.0e4

408.10 104.20

60

80

100

120

143.08

140

160

180

200

220

240 260 m/z, amu

280

300

320

340

360

380

400

420

440

110.1

4.4e6 4.2e6 4.0e6 3.8e6 3.6e6 3.4e6

Intensity (cps)

CH3

O

H+ NH2 HO

3.2e6 3.0e6 2.8e6 2.6e6 2.4e6 2.2e6 2.0e6

H+

(Product ion m/z, 110.1)

NH2 O H+

(Product ion m/z, 138.2)

H N

138.2

O

O

1.8e6 1.6e6 1.4e6 1.2e6 1.0e6 8.0e5 6.0e5 4.0e5 2.0e5

(Parent ion m/z, 180.1) 180.1

93.1 42.9

20

30

40

82.1

65.1

50

60

70

80

93.9

90

152.1 108.9 111.2

100

110 120 m/z, amu

134.2

130

162.2

140

150

160

170

180

190

200

Figure 1. Product ion spectra of (A) miltefosine (m/z 408.1), and (B) phenacetin (m/z 180.1).

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Research Article  Jaiswal, Sharma, Shukla & Lal

CS0 – phenacetin (IS) (Unknown) 180.100/138.200 Da – sample 1 of 12 from CS.wiff Area: 54400. counts Height: 5140. cps RT: 1.57 min

CS0 – 408.1 / 125.1 (Unknown) 408.100/125.100 Da – sample 1 of 12 from CS.wiff (peak not found) 3.36

1.55

75

240

70

220

65

200

60

1.44

45 40 35 30

0.58 1.04

1.49

1.97 2.32 1.72

2.78 3.61

4.16

4.56

3.04

0.12

3.94

Intensity (cps)

Intensity (cps)

50

160

2.38

1.63

0.72

4.85

2.86

3.80

1.90

0.43

3.41

3.58

4.06 4.36

140 120 100

25

80

20

60

15

40

10

20

5 0

0 0.5

1.0

1.5

2.0 2.5 3.0 Time (min)

3.5

4.0

0.5

4.5

CS0 IS – 408.1 / 125.1 (Unknown) 408.100/125.100 Da – sample 1 of 12 from CS.wiff (peak not found)

1.0

1.5

2.0

2.5 3.0 Time (min)

3.5

4.0

4.5

CS0 IS – phenacetin (IS) (Unknown) 180.100/138.200 Da – sample 1 of 12 from CS.wiff Area: 54400. counts Height: 5140. cps RT: 1.57 min 4.88

50

1.57

5000

0.54

45

4500

40

1.69

3.37

0.19

3.94

4000

1.61

30

3.58

4.14

4.57

1.56 1.22

25

2.90 1.17

1.90

20

2.77 2.00 0.69

15

2.10

Intensity (cps)

4.40

35 Intensity (cps)

1.03

180

4.74

55

0.15

3500 3000 2500 2000 1500

10

1000

5

500

0

0 0.5

1.0

1.5

2.0 2.5 3.0 Time (min)

3.5

4.0

4.5

0.5

1.0

1.5

2.0 2.5 3.0 Time (min)

3.5

4.0

4.5

Figure 2. Representativemultiple reaction monitoring chromatogram of (A) drug-free plasma, (B) hamster plasma spiked with internal standard (IS) (10 ng/ml) only, (C) hamster plasma containing miltefosine (2.5 ng/ml) and IS (10 ng/ml) and (D) test plasma sample taken at 2 h from a hamster treated with 10 mg/kg oral dose of miltefosine (see facing page for [C] and [D]).

10.4155/bio.16.7

Bioanalysis (Epub ahead of print)

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LC-coupled ESI MS for quantification of miltefosine in human & hamster plasma

CS1 - phenacetin (IS) (Standard) 180.100/138.200 Da - sample 3 of 12 from CS.wiff Area: 51200. counts Height: 4880. cps RT: 1.56 min 1.57

CS1 - 408.1 / 125.1 (standard) 408.100/125.100 Da - sample 3 of 12 from CS.wiff Area: 897. counts Height: 53.7 cps RT: 1.77 min

4500

450

1.79

4000

400

3500 Intensity (cps)

Intensity (cps)

350 300 250 200

2500 2000

1000

100

0

3000

1500

150

50

2.20 2.54 2.82 3.18

0.46

0.5 1.0

1.5

2.0

2.5 3.0

500 3.65

3.5

4.24

4.0

4.77

0

4.5

0.5 1.0

1.5

Time (min)

3.5

4.0

4.5

1.57

1.78

4000 3500 3000 Intensity (cps)

Intensity (cps)

2.0 2.5 3.0 Time (min)

2 HR H - phenacetin (IS) (Unknown) 180.100/138.200 Da - sample 10 of 50 from TEST SAMPLE.wit Area: 42400. counts Height: 4210. cps RT: 1.57 min

2 HR H - 408.1 / 125.1 (Unknown) 408.100/125.100 Da - sample 10 of 50 from TEST SAM… Area: 71300. counts Height: 3390. cps RT: 1.78 min

3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0

Research Article

2500 2000 1500 1000 500

0.5 1.0

1.5

2.0 2.5 3.0 Time (min)

3.5

4.0

= 0.996, where y and x represent the A Miltefosine /A IS and concentration of analyte in ng/ml, respectively. The intra-assay and inter-assay accuracy and precision of four different QCs (LLOQ, LQC, MQC and HQC, each n = 6) were found within acceptable limits (85–115%, [14]) and are summarized in Table 3. Intraassay precision, as indicated by %RSD, varied from 2.79 to 11.81. However, inter-assay precision evaluated at the same concentrations was ≤9.07. The accuracy of the method (%bias) ranged from -14.20 to 10.79.

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4.5

0

0.5

1.0

1.5

2.0 2.5 3.0 Time (min)

3.5

4.0

4.5

Additionally, three sets of calibration with QC samples were prepared, processed and analyzed by three independent analysts for the assurance of method. A 100- and 200-fold dilution of DIQC samples by blank drug-free plasma (hamster and human) prior to extraction was used for determining dilution integrity. Sextuplicates of DIQC samples were extracted and analyzed along with one of the validation batches. The accuracy (%bias) and precision (%RSD) of DIQC samples varied from -11.67 to 13.33 and 4.59 to 5.36,

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10.4155/bio.16.7

Research Article  Jaiswal, Sharma, Shukla & Lal

Table 3. Accuracy (%bias) and precision (%RSD) for miltefosine in hamster and human plasma (n = 6 at each concentration). Concentration (ng/ml)

LLOQ

LQC

MQC

HQC

 

2.50

3.75

180.00

360.00

%BiasIntra-assay

10.79

1.35

-3.07

3.26

%BiasInter-assay

6.81

6.17

-1.00

4.64

%RSDIntra-assay

10.64

9.54

11.81

8.32

%RSDInter-assay

5.62

5.49

9.07

7.60

Mean ± SD

104.90 ± 7.65

105.70 ± 8.67

102.10 ± 11.21

97.80 ± 7.51

%CV

7.29

8.21

10.98

7.86

Mean ± SD

105.51 ± 7.03

104.46 ± 6.13

103.48 ± 11.69

98.30 ± 7.34

%CV 

6.66

5.87

11.30

7.47

Hamster plasma

Combined recovery:

True recovery:

Human plasma %BiasIntra-assay

-5.78

5.14

-2.43

3.19

%BiasInter-assay

-14.20

6.14

-2.43

3

%RSDIntra-assay

8.23

8.39

2.79

6.24

%RSDInter-assay

6.57

6.26

2.81

5.39

Mean ± SD

97.40 ± 9.35

105.30 ± 7.86

96.70 ± 2.87

101.50 ± 5.78

%CV

9.60

7.46

2.90

5.70

Mean ± SD

98.1 ± 9.62

105.35 ± 3.33

97.28 ± 4.56

101.91 ± 5.93

 %CV

9.81

2.00

4.56

5.82

Combined recovery:

True recovery:

respectively, demonstrating that the samples with concentrations greater than the ULOQ can be diluted (up to 200-fold) and analyzed to obtain precisely accurate results. The mean true extraction recovery of miltefosine from hamster and human plasma was >98.30% and >97.28%, respectively. The recovery of miltefosine from plasma was consistent, accurate, precise and concentration-independent (Table 3) . The matrix effect for the analyte (3.75, 180 and 360 ng/ml) and IS (10 ng/ml) in hamster and human plasma from six different lots was less than ±15%, indicating that protein precipitation method is suitable with negligible ion suppression or enhancement of miltefosine by endogenous matrix of human and hamster plasma. This was further confirmed by postcolumn infusion technique. It was observed that endogenous matrix impurities eluted from 0.6 to 1 min leading to suppression of the analyte signal during this interval. The elution of IS and miltefosine starts after 1.3 min (retention times 1.6 and 1.8 min for IS and analyte, respectively). Following this, chromatography was continued for another

10.4155/bio.16.7

Bioanalysis (Epub ahead of print)

2 min before next injection, which did not show any suppression or enhancement due to endogenous impurities. This indicated that amide column successfully separated majority of the endogenous matrix impurities from analytes. Aqueous ACN (50%, v/v) was used as rinsing solution to minimize carryover effect, if any. Analysis of processed double blank sample after ULOQ showed that the peak area of the eluting peak at the retention time of the miltefosine and IS was less than 5% of the respective peak areas at LLOQ indicating an insignificant carryover effect. An array of stability studies of miltefosine was performed as described above and the results are presented in Table 4. Miltefosine was found to be chemically stable in stock solutions (SS and WS) on storage at 4°C (stored in refrigerator) for 45 days as the difference between the peak responses of miltefosine in analytical standards (at 3.75, 180, 360 ng/ml) prepared from fresh and stored stock solution was found to be 4.00 ± 0.02%. The accuracy of stock solution stability samples was between 96.40 and 99.75% with

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LC-coupled ESI MS for quantification of miltefosine in human & hamster plasma

Research Article

Table 4. Stability of miltefosine under different storage conditions (n = 6 at each concentration). Storage conditions  

Bench-top stability (room temperature, 6 h)

Nominal Hamster plasma concentration Measured mean %CV Accuracy (ng/ml) concentration (%) (ng/ml)

Measured mean concentration (ng/ml)

%CV

Accuracy (%)

3.75

3.39 ± 0.21

6.20

90.39

3.52 ± 0.32

9.01

93.29

180.00

173.60 ± 8.39

4.83

96.50

170.50 ± 8.58

5.03

94.67

360.00 Autosampler stability (4°C, 24 h) 3.75 180.00

Human plasma

370.83 ± 24.82

6.69

104.80

331.75 ± 22.03

6.82

92.12

3.35 ± 0.10

2.99

89.33

3.40 ± 0.20

5.88

90.67

160.75 ± 7.80

4.86

89.31

161.50 ± 5.51

3.41

89.72

360.00

328.33 ± 12.58

3.83

91.20

320.00 ± 10.80

3.38

88.89

Long-term stability (-80°C, 30 days)

3.75

3.49 ± 0.48

11.79

92.83

3.67 ± 0.46

12.60

97.91

180.00

163.60 ± 11.39

6.96

90.86

172 ± 7.64

4.49

95.63

360.00

346.83 ± 14.45

4.17

96.90

337.4 ± 20.08

5.95

93.70

Freeze–thaw stability (second cycle; -80°C to room temperature)

3.75

3.53 ± 0.36

10.28

94.13

3.60 ± 0.35

9.65

95.81

180.00

177.83 ± 11.92

6.70

98.67

179.17 ± 9.75

5.44

99.53

360.00

347.67 ± 14.07

4.05

96.58

350.2 ± 9.00

2.57

97.30

PK study

The successfully validated LC–MS/MS method was applied for the quantification of miltefosine after 10 mg/kg per oral dose in hamster. The PK studies revealed that the animals tolerated the treatment as no peculiarities in their behavior were observed. The drug was quantified in test plasma samples up to 408 h postdosing. The plasma concentration–time profile of miltefosine following 10 mg/kg single oral dose in hamster is shown in Figure 3. The plasma concentration–time data were subjected to noncompartmental approach using Phoenix WinNonlin computer software to calculate the PK parameters, which are listed in Table 5. Following single oral dose, the Cmax (29.19 ± 2.45 μg/ml) of miltefosine was achieved at 10 h in all the three hamsters demonstrating slow absorption. Also, miltefosine exhibited slow elimination with a long-terminal half-life (94.42 ± 4.75 h). The volume of distribution over bioavailability (Vd /F) of miltefosine (0.63 ± 0.03 l/kg) is higher than the total blood volume (0.09 l/kg; [17]) indicating extravascular distribution of drug. The clearance over bioavailability

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of miltefosine (4.69 ± 0.33 ml/h/kg) is lower than the hepatic blood flow (6.50 ml/min; [18]) of hamster indication negligible extra hepatic elimination. Conclusion Miltefosine, an alkylphosphocholine compound, is the first and still the only oral drug available for treatment of visceral, cutaneous and mucosal leishmaniasis [19,20] . A rapid and simple LC–MS/MS method for quantification of miltefosine in hamster and human plasma is developed and appropriately validated over the concentration range of 2.5–400 ng/ml using the FDA guidelines for bioanalysis. The method has short analysis time (

LC-coupled ESI MS for quantification of miltefosine in human and hamster plasma.

Leishmaniasis, a fatal parasitic disease, is the second largest parasitic killer in the world and miltefosine is the first and only oral drug availabl...
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