1. INTRODUCTION:

Leishmaniasis is a protozoan disease caused by a Leishmania type parasite spread by certain sandflies¹. Hexadecylphosphocholine is a lipid-like product that is active on the cell membrane and interferes with cell growth and differentiation of breast cancer. In India, since 1998, it has been used to treat visceral leishmaniasis². Miltefosine is a broad-spectrum antimicrobial, anti-leishmanial, anti-cancer phospholipid drug³.

It is mainly an approved oral and topical dosage form used to treat visceral (caused by Leishmania donovani), cutaneous (caused by Leishmania braziliensis, Leishmania guyanensis, and Leishmaina panamensis), and mucosal conditions (caused by Leishmania braziliensis) of leishmaniasis⁴. Miltefosine is used as a drug for first-line treatment of primary amebic meningoencephalitis and granulomatous amebic encephalitis caused by free-living amebae⁵. In Florida, the USA, meningoencephalitis caused by brain eater Ameba was successfully treated by miltefosine^6,7. Leishmania donovani is highly susceptible to miltefosine, and this drug is also active against neoplastic cells because miltefosine affects apoptosis and lipid-dependent cell signalling pathway⁶. Miltefosine affects macrophage's lipid homeostasis, autophagy, and NLRP3 inflammasome activity⁸. NOD is an intracellular sensor of pathogen-associated molecular patterns that enter the cell via phagocytosis or pores and damage-associated molecular patterns associated with cell stress⁹. Miltefosine increases phosphorylation of kinases such as AMPK, ULK1 and inhibits the TLR signalling pathway, reduces LPS induced pro-IL-1β mRNA levels, and reduces the release of IL-1β¹⁰. It also protected mitochondrial membrane potential by reducing endotoxin mediated mitochondrial reactive oxygen species. Miltefosine inhibits mitochondria's cytochrome –c oxidase, leading to mitochondrial dysfunction and apoptosis-like cell death¹¹. Antineoplastic and antileishmanial mechanism of action of miltefosine represented in Figure: 1, relatively same which include inhibition of phosphotidylcholine biosynthesis and Akt which is known as protein kinase B, which is a crucial protein within the PI3K/Akt/mTOR intracellular signaling pathway that pathway involved in regulating the cell cycle¹². Miltefosine is also effective against Trypanosome cruzi which is responsible for Chagas disease, and metronidazole resistant styrains of Trichonomas vaginalis. It has broad-spectrum antifungal activity. Miltefosine is an effective palliative option for cutaneous metastasis for breast cancer (response rate of 42%). An antibiotic composition comprising miltefosine and polyhexamethylene biguanide use in the prevention or treatment of a condition of the eye or the skin is provided by the present invention (Patent 2013WO2013135571A1). Miltefosine is used as a topical agent for dressing fungating wounds¹³.

Therefore, in the present study, efforts were given to develop and validate an analytical method for estimating miltefosine in dissolution sample, solubility sample, and in force, degradation sample also used for impurity analysis by LC-MS/MS.

Hence, the scope of the present work is to develop a fast, rapid, sensitive, and specific method of LC-MS/MS with a short time-consuming method and gives better sensitivity.

2. MATERIAL AND METHODS:

2.1 Chemical and Reagents:

Methanol was purchased from J T Bakar, and isopropyl alcohol, ammonia solution, chloroform was AR grade, and usable solvents were HPLC grade. Milli-Q water used in the entire analysis was prepared from Millipore (Elix, Milli-Q A10 Academic, Bedford, MA, USA) until conductivity less than 0.05µs/cm was achieved.

2.2 API sample Source:

The source of Miltefosine (API) is Albert David Limited, Block-D, 3^rd Floor, Gillander House, 8, Netaji Subhas Road, Kolkata-700001, India

2.3 Method Development And Gradation Technique For Detection And Quantification Of Miltefosine By LC-MS/MS:

Miltefosine is a phospholipid compound, which chemical formula is C₂₁H₄₆NO₄P, and the chemical name of miltefosine is hexadecyl 2-(trimethylazaniumyl) ethyl phosphate [Figure: 2] and molecular weight 407.58 and monoisotropic molecular weight 407.3164. For quantitation used positive ionization mode was selected in the present study to fragment the analyte and IS to obtain intense and consistent product ions. The precursor ions (protonated) [M+H]⁺at m/z 408.1(highest peak), 391.2(2^nd peak), 430.3(3^rd peak), 446.1(4^th peak) were observed in Q1 MS scanning for miltefosine, and characteristic product ions or fragment ions found in Q3 MS scanning were at m/z 124.9, 184.2, 166.0. However, the most stable and consistent fragment ion selected was m/z 124.9[(M-C₁₉H₄₂N+H)]⁺ by releasing hexadecyltrimethylamine, ethyl phosphate organophosphate oxoanion and has a role as an epitope. [Figure: 3]. Metoprolol is used as an internal standard (IS). The precursor ions (protonated) [M+H]⁺at m/z 268.2(highest peak) 269.3(2^ndpeak), 241.2(3^rdpeak), 22.9(4^thpeak))were observed in Q1 MS for metoprolol and characteristic product ions or fragment ions found in Q3 MS was m/z 116.2, 159.3, 133.1, 74.1, 98.0, 121.2. However, the most stable and consistent fragment ion selected was m/z 116.2 for five consecutive rings.

This gradient method was performed on a Shimadzu HPLC system equipped with an LC-20AD binary pump, SIL-20A autosampler, CTO-10ASvp Oven, and CBM-20A lite system control compartment. In addition, mass spectrometric detection of miltefosine was performed on an API 2000 triple quadrupole mass spectrometer (Applied Biosystems/MDS SCIEX, Toronto, ON, Canada) equipped with a Turbo electrospray ionization (ESI) interface. The total mass parameters for both miltefosine and IS were elaborated in Table: 1.

The chromatographic elution of miltefosine on a Phenomenex Kinetex 5µ C18 100A 50*3mm column was initiated as a rapid, sensitive, and rugged analytical method covering the dynamic linear range. The final solvent used for analysis was 10mM ammonia solution in methanol as an organic solvent and 30Mm ammonium acetate buffer with 0.069% (v/v) ammonia solution and Milli Q water having apparent pH 8.90 at a flow rate of 0.6000mL/min. No additional peaks were observed in one report when the column was used even under MRM mode. The chromatographic elution time for miltefosine and IS (metoprolol) was 3.45 and 2.04 min, respectively, in a run time of 6.00 min. This analysis was done by the gradation method in which 0.01min to 0.50 min organic solvent 10% and then 0.5 min to 2.50min organic solvent 90% and then from 2.50 min aqueous solvent 90% run-up to 6.00 min for washing purpose. The gradient curve of method development is shown in Figure 4. The MRM chromatogram of miltefosine and IS (metoprolol) is shown in Figure 5. a.

2.4 Sample preparation procedure:

2.4.1 Stock solution preparation:

Weighed about 5mg of miltefosine and dissolved in 5ml chloroform and mixed well and sonicated and stored in the refrigerator at 2-8°C, which was used for a final concentration of miltefosine. Weighed about 5mg of metoprolol and dissolved in 5ml DMSO and mixed well and sonicated and stored in the refrigerator at 2-8°C, which was used to prepare the final concentration.

2.4.2 Calibration standards preparation:

Stock solutions of miltefosine and IS (metoprolol) were prepared to obtain 1mg/ml concentrations. The stock solutions were then gradually diluted with methanol: water: 50:50 (v/v) to obtain calibration samples of 125, 250, 500, 1000, 2000, and 4000ng/ml for miltefosine.

3. METHOD VALIDATION:^14-16

The method validation was conducted following the guidelines US-FDA and EMA for selectivity, sensitivity, linearity, precision, accuracy, and stability.

3.1 Specificity, selectivity, and linearity:

Specificity and selectivity are the main features of an analytical method to quantify and differentiate all analyte from a complex mixture. A chromatogram of LLOQ determines this. Linearity is illustrated by the nominal percentage of calibration concentrations of three consecutive days. The calibration curve should be linear, and the regression value should not be more than one.

3.2 Precision and accuracy:

Precision and accuracy are two types- one is inter-day, and the other is intraday precision and accuracy. All three quality control concentrations should be within ±15% of the specified concentrations, except for the LLOQ within ±20%. But precision is determined by % CV, which should be within15% for all quality control concentrations.

3.3 Stability:

According to guidelines, short term and bench to stability should be within 90-110%; on the other hand, freeze-thaw and autosampler stability should be within 85-115%

4. RESULT AND DISCUSSION OF METHOD VALIDATION: ^14,15,16

4.1. Specificity, selectivity, and linearity:

Following calibration standards (ng/ml) were prepared- 125, 250, 500, 1000, 2000, and 4000ng/ml for miltefosine. The proposed assay was found linear. The representative calibration curves were shown as linearity graphs in Figure-5.b. Back calculated a concentration of the calibrant samples of the linearities for miltefosine was also represented in Table-2. The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) were found at 1.45ng/ml and 125ng/ml, respectively, for miltefosine.

Figure-1: Antileishmanial mechanism of action of miltefosine

Figure-2: Chemical structure of miltefosine

Figure-3: Parent ion and product ion scan of miltefosine

Figure-4: Gradient curve of miltefosine

Figure-5.a: MRM chromatogram of miltefosine and IS

Figure-5.b: Calibration curve of miltefosine

4.2. Precision and accuracy:

Between–run precision values (%CV) ranged from 2.515% to 6.736% for miltefosine. Between–run accuracy values (% nominal) were 109.276% for LLOQ, 97.493% for low QC (LQC), 96.866% for medium QC (MQC), and 96.544% for high QC (HQC) samples.

Within-run precision values (%CV) ranged from 1.852% to 7.599% for miltefosine. Within-run accuracy values (% nominal) were 111.298% LLOQ, 96.743% for low QC (LQC), 99.305% for medium QC (MQC) and 98.276% for high QC (HQC) samples. The between run and within run precision results were represented in Table-3. R square values were 0.9842, 0.9933, 0.9944 for Linearity 1, 2, and 3 respectively. The mean R square value was 0.990633333 and %CV 0.565144693. The result is tabulated in Table-3.

4.3. Stability:

The stability study data were elaborated in Table-4.

Benchtop: Qc samples were kept for 24 hrs at room temperature and then processed and analyzed. % Stability was within 90.00% to 95.52% for miltefosine. So the values are acceptable.

Autosampler stability:

The Auto Sampler Stability of miltefosine ranges between 89.72% to 98.54% after three cycles. So the values are acceptable.

Freeze-thaw stability:

The stability of low, medium, and high-quality control samples were determined after three freeze-thaw cycles compared against freshly thawed samples of the same concentration. The stability of miltefosine ranges between 90.71% - 97.99% after three cycles. So the values are acceptable.

Short term stability: The Percentage of short term stability was within 92.04% to 98.02% for miltefosine. So the values are acceptable.

Table-1: Optimized instrumental (mass) parameters for the analyte and IS

Parameter (s)	Value
Ionization mode	MRM (+ve)
Source temperature (ºC)	400
Dwell time per transition (msec)	100
Curtain gas (psi)	30
CAD gas (psi)	6
Ion spray voltage (V)	5500.00
Ion source gas 1 (psi)	55
Ion source gas 2 (psi)	45
Focusing potential (V)	400
Declustering potential (V)	38 (Miltefosine) and 25 (IS)
Entrance potential (V)	11
Collision energy (V)	45 ( Miltefosine ) and 30 (IS)
Collision cell exit potential (V)	6 (analytes and IS)
Transition pair of Miltefosine (analyte)	408.1/124.9
Transition pair of Metoprolol (IS)	268.2/116.2

Table-2: Pre-study linearity of detector response

Linearity	Concentration(ng/ml)						Statistics
	125	250	500	1000	2000	4000	Slope(m)	Intercept(c)	R²
LIN 1	138.82	211.29	428.08	918.85	2291.73	4493.64	0.00138	0.177	0.9842
LIN 2	133.04	216.25	476.51	1127.77	1953.07	4053.36	0.000235	0.00477	0.9933
LIN 3	127.27	254.90	458.96	904.77	2008.03	4542.26	0.000241	0.000519	0.9944
Avg.	133.043	227.480	454.517	983.797	2084.277	4363.087	0.000618667		0.990633333
± SD	5.775	23.876	24.519	124.883	181.749	269.331	0.000659341		0.005598512
% CV	4.341	10.496	5.394	12.694	8.720	6.173	106.5745		0.565144693
%Nominal	106.435	90.992	90.903	98.380	104.214	109.077
Linearity	Concentration(ng/ml)						Statistics
	125	250	500	1000	2000	4000	Slope(m)	Intercept(c)	R²
LIN 1	138.82	211.29	428.08	918.85	2291.73	4493.64	0.00138	0.177	0.9842
LIN 2	133.04	216.25	476.51	1127.77	1953.07	4053.36	0.000235	0.00477	0.9933
LIN 3	127.27	254.90	458.96	904.77	2008.03	4542.26	0.000241	0.000519	0.9944
Avg.	133.043	227.480	454.517	983.797	2084.277	4363.087	0.000618667		0.990633333
± SD	5.775	23.876	24.519	124.883	181.749	269.331	0.000659341		0.005598512
% CV	4.341	10.496	5.394	12.694	8.720	6.173	106.5745		0.565144693
%Nominal	106.435	90.992	90.903	98.380	104.214	109.077

Table-3: Precision and Accuracy (n=5)

	Between run			Within run
	Mean ± SD	C.V.%	Absolute bias (%)	Mean ± SD	C.V.%	Absolute bias (%)
LLOQ (125ng/ml)	136.595±7.538	5.518	109.276	139.122±6.971	5.010	111.298
LQC (375 ng/ml)	365.597±24.628	6.736	97.493	362.786±27.567	7.599	96.743
MQC (1500ng/ml)	1452.993±56.693	4.108	96.866	1489.582±57.496	3.860	99.305
HQC (3000ng/ml)	2896.328±72.844	2.515	96.544	2948.270±54.593	1.852	98.276

Table-4: Stability Study (Freeze-thaw, Short term, Autosampler, Benchtop stability)

		Inj No.	LQC (375 ng/ml)	MQC (1500 ng/ml)	HQC (3000 ng/ml)
	Freshly Thawed	1	390.69	1430.61	2935.76
		2	369.11	1491.50	2943.68
		3	389.85	1484.91	3055.34
		4	401.89	1363.41	3115.07
		5	388.07	1407.88	3335.11
		Mean	387.92	1435.66	3076.99
Freeze-thaw stability	After 3 cycle	1	354.86	1388.20	2863.89
		2	368.54	1382.65	2822.15
		3	348.81	1390.94	2820.65
		4	349.99	1386.33	2780.15
		5	337.23	1485.7	2768.87
		Mean	351.89	1406.76	2811.14
	% Stability		90.71	97.99	91.36
Short term stability	After 24 hours.	1	356.69	1441.52	2818.12
		2	356.83	1448.11	2720.22
		3	359.15	1354.48	2785.84
		4	347.92	1382.08	2948.93
		5	371.76	1410.27	2886.52
		Mean	358.47	1407.29	2831.93
	% Stability		92.41	98.02	92.04
Autosampler stability	After 24 hours in auto sampler (15ºc)	1	347.37	1398.54	2897.23
		2	338.90	1409.95	3061.68
		3	339.84	1410.81	2923.77
		4	348.23	1404.54	2943.09
		5	365.92	1449.32	2884.49
		Mean	348.05	1414.63	2942.05
	% Stability		89.72	98.54	95.61
Benchtop stability	After 24 hours in laboratory room temperature	1	340.10	1369.81	2869.76
		2	346.85	1388.88	2902.22
		3	352.40	1345.29	2797.22
		4	357.09	1382.82	2886.09
		5	349.26	1369.74	2965.60
		Mean	349.14	1371.31	2884.18
	% Stability		90.00	95.52	93.73

5. CONCLUSION:

Miltefosine is an essential phospholipids drug in breast cancer, leishmaniasis, cutaneous cancer, meninzoencephalitis, antimicrobial, etc. Aimed of this study was to develop a validated analytical method for the detection and quantification of miltefosine from analytical samples. So this developed method was validated as per USFDA and EMA guidelines. Miltefosine is generally a weakly acidic drug, and better ionization was done in a strongly basic solution and retention of this drug at 3.45 min. in basic pH. But if the elution of this drug was carried out in the isocratic method and acidic P^H solvent, the response found was significantly less. So, in the present study, a gradient method was applied: the short run time (6min.) and only very few minutes (2.5min.) was used 90% organic solvent at 0.6ml/min flow rate was.

Therefore, it can be concluded that this analytical method of miltefosine was developed using LC-MS/MS (API-2000) by applying significantly less organic solvent and concise run time that is time-consuming and much better than other HPLC methods. This method was simple, specific, sensitive, and reproducible, fully validated as per USFDA guidelines. Therefore this analytical method was more superior to be applied to dissolution studies, solubility studies, force degradation studies, and impurity analysis of miltefosine by LC-MS/MS (API-2000)

6. CONFLICT OF INTEREST:

In the future, this validated analytical method will be used to estimate miltefosine in dissolution sample, solubility sample, and in force, degradation sample also used for impurity analysis by LC-MS/MS.

7. ACKNOWLEDGEMENT:

The authors are also thankful to Albert David Limited, Block-D, 3^rd Floor, Gillander House, 8, Netaji Subhas Road, Kolkata-700001, India, for providing the necessary API at TAAB Biostudy Services, Kolkata-700032, India. In addition, the authors are also thankful to M/S, TAAB Biostudy Services, Kolkata-700032, India, National Institute of Technology, Tiruchirappalli, Government of India, India and Bioequivalence Study Centre, Jadavpur University, Kolkata-700032, India, for providing the necessary instrumental facilities.

8. DECLARATIONS:

Ethics approval and consent to participate:

This article does not involve human participants, so it is not applicable. Miltefosine is an FDA approved drug for leishmaniasis, initially investigated as an anti-cancer agent. A topical formulation of miltefosine was studied to treat skin metastases from breast cancer and was eventually approved for this indication in several countries. WHO/TDR (TDR: Special Program for Research and Training in Tropical Diseases of UNDP/World Bank/WHO) set up a study program to jointly develop miltefosine as an oral treatment for VL. During the progress of this work, the study program and its progress were also discussed with the Indian Council of Medical Research (ICMR). The first dose-ranging trial (pilot study 0033) was sponsored by ASTA Medica/Zentaris and investigated doses between 50mg every other day and 250mg/day for four weeks in 5 patients per treatment group. The lower doses (50mg every other day and 100mg every other day) were not effective; the higher doses (200mg per day and 250mg per day) were poorly tolerated. However, the moderate doses (100mg per day and 150mg per day) appeared effective and tolerated. This miltefosine is now marketed for VL (and CL) in 14 countries worldwide. This miltefosine treats visceral leishmaniasis; all study data are already included in NDA 204684.

This formulation of miltefosine, which is structurally and chemically, therapeutically the same as previous miltefosine drug, is new for Albert David Limited, Kolkata, India. So, this proposed analytical method was performed by active pharmaceutical ingredients (API) used in an organic solvent to identify and quantify miltefosine in force degradation study samples, solubility study samples, etc. This will be used for the new formulation development of miltefosine. The article also follows the guidelines laid by the National Institute of Technology, Tiruchirappalli, Tamil Nadu, India

Safety Guidelines:

The efficacy and safety of miltefosine in treating visceral leishmaniasis had been investigated in prospective phase-1-3 clinical trials involving Indian patients. Oral use of miltefosine in cancer patients was associated with gastrointestinal side effects, preventing the long-term administration of therapeutically active dosages in this patient population. In addition, daily doses of 150mg and higher were often associated with dose-limiting side effects, including nausea and vomiting and loss of appetite.

Consent for publication:

Not applicable. However, the authors declare that there is no known competing financial interest or personal relationships that could have appeared to influence the work reported in this manuscript.

Availability of data and material:

Apart from all data generated and analyzed are available with this manuscript.

Competing Interests:

The authors declare that they have no competing interests.

9 AUTHORS' CONTRIBUTIONS:

Pallab Mandal: Conceptualization, Data curation, Formal analysis, Investigation, Validation, writing the draft manuscript, Pradip Kundu: Conceptualization, Data curation, Formal analysis, Investigation and drug information, Soumya Chakraborty: Conceptualization, Data curation, Formal analysis, Investigation, Validation, writing the draft manuscript, RakeshBera: Conceptualization, Data curation, Formal analysis, Investigation, Validation, writing the draft manuscript, Nilendra Chatterjee: Conceptualization, Data curation, Formal analysis, Investigation, Validation, writing the draft manuscript, Sourav Poddar: Conceptualization, Data curation, Formal analysis, Investigation, Software utilization, Validation, Visualization, and Writing, review and editing – original and final manuscript, D.P. Ghosh: Conceptualization, Data curation, Formal analysis, Investigation, Software utilization, Validation, Visualization, and Writing, review and editing – original and final manuscript, Tapan Kumar Pal: Conceptualization, Data curation, Formal analysis, Investigation, Software utilization, Validation, Visualization, and Writing, review and editing – original and final manuscript.

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