DETAILS

DBA Intermediates of Lumefantrine | Manufacturers | Suppliers | India

DBA Intermediates of Lumefantrine | Manufacturers | Suppliers | India

The list of Lumefantrine intermediates that we offer can be found below. Please click on the intermediate name to see additional information about the intermediate you desire. Let’s say you can’t find the Lumefantrine intermediate you need on this website. In such case, email us a sourcing request for the Lumefantrine intermediate you’re seeking for, along with its Cas No and Intermediate name, and we’ll get back to you with further information as soon as possible. To send an email, please click here.

Please feel free to contact us if you require any upstream or downstream intermediates for your Intermediates of Lumefantrine requirement. We will find the right manufacturer, supplier, or principals according on your specific needs, who are dependable and match your quality standards.

INTRODUCTION

Primaquine (PQ) diphosphate is a prototype antimalarial medication in the 8-aminoquinoline class. It is the only medicine on the market that prevents relapses of infections caused by the reactivation of dormant forms of the malaria parasites Plasmodium vivax or Plasmodium ovale (also known as hypnozoites) in the liver. PQ is also effective against all developing malaria parasites in the liver and mature P. falciparum gametocytes in the blood. Despite its poor action against asexual blood stages of P. falciparum and other malaria parasites, PQ is the only medicine capable of substantially treating P. ovale and P. malariae when taken in combination with a schizonticide agent.

Infections with vivax Despite its unique role in the physician’s arsenal for treating malaria, PQ’s usage in clinical practise is restricted since it (together with pamaquine, tafenoquine, and a few other medications) can induce severe hemolytic anaemia in people with glucose-6-phosphate dehydrogenase deficiency. PQ can also cause methemoglobinemia, but this toxicity seldom causes noticeable symptoms and is usually self-limiting.

Furthermore, the therapeutic response to PQ is dosage and drug-metabolizing enzyme phenotype dependent. PQ is a pro-drug transformed by CYP2D6 enzymes into oxidised metabolites responsible for activity against dormant forms of P. ovale and P. vivax in the liver, according to investigations conducted over the last ten years. In human populations, the CYP2D6 enzyme is highly polymorphic, and patients with a CYP2D6 poor metabolizer phenotype do not react to PQ therapy. To cure malaria, the PQ dose must also be changed in overweight people.

As a result, sensitive and reliable methods for analysing PQ in blood plasma are required for monitoring medication levels in patients receiving therapy and studying the kinetics of this antimalarial agent in animal models.

DBA Intermediates

Table 1 highlights the key features of HPLC techniques for the determination of PQ in biological matrices (such as whole blood, serum, plasma, liver tissues, and blood cells) that have been described to date.

The majority of PQ analysis methods in plasma use a liquid-liquid extraction approach followed by protein precipitation (PP), although some also use solid phase extraction (SPE) or a mix of liquid-liquid extraction and SPE procedures. When PP and SPE procedures are used, PQ recovery rates are often higher. The mobile phases varies among available methods, as indicated in.

Organic modifiers are frequently added to a mixture of organic solvents (methanol or acetonitrile) with water (acid/basic or neutral) or a buffer solution. The most frequent mode of elution is isocratic, however some procedures use the more time-consuming gradient mode. Most methods use reversed phase C18 columns with various lengths, internal diameters, and particle sizes as the stationary phase, while a few others use silica-modified stationary phases like phenyl and cyanopropyl.

All approaches necessitate large sample quantities (500L of plasma), which limits their application in investigations with small rodents like mice and hamsters. PQ’s long retention durations mean that these chromatographic procedures will take a long time to analyse (8 minutes). Furthermore, a DAD-UV detector has high detection and quantification limits.

We designed and validated a rapid, sensitive, cost-effective, and robust HPLC-DAD-UV approach for analysis of PQ in blood plasma due to the limitations of existing methods. Because this approach necessitates small plasma volumes, it is particularly well suited to studies of PQ kinetics in small rodents.

METHODS
Reagents

All of the compounds were analytical reagent grade or purer. Tedia (Rio de Janeiro, Brazil) provided HPLC-grade methanol and acetonitrile, and Lichrosolv ultrapure acetonitrile was used to test robustness (CAS 75-05-8; Merck Millipore, Darmstadt, Germany). Merck Millipore provided ammonium acetate (CAS 631-61-8, CH3COONH4), zinc sulphate heptahydrate (ZnSO4•7H2O), and acetic acid (CAS 64-19-7, CH3COOH).

Genix Indstria Farmacêutica Ltda (PP3016PQRJ, 98-102 percent; Anápolis, Brazil) provided the PQ diphosphate standard. Hipolabor provided sodium heparin (5,000IU/mL-1) for this study (Belo Horizonte, Brazil). A Milli-Q® purifying water system delivered ultrapure water on a daily basis (Merck-Millipore).

DBA Intermediates

Solutions

PQ diphosphate aqueous solutions were made by dissolving 17.6mg of the salt in ultrapure water to a final volume of 50mL, equating to 200g.mL-1 PQ base. Stock solutions were held at -20°C in amber flasks. PQ is stable for at least 7 days in this storage circumstances (as determined by the signal area of a standard solution in an HPLC-DAD-UV analysis). The working solutions were made via repeated dilution of the stock solution on a regular basis. 0.77g ammonium acetate was dissolved in 1L ultrapure water to make ammonium acetate buffer (10mM). A 10% w/v acetic acid solution was used to modify the pH of the solution to 3.8. [DBA Intermediates]

Animals

Blood plasma samples from female DBA-2 mice were used to perform quantitative PQ tests utilising the novel approach. The Oswaldo Cruz Foundation Central Animal House (CECAL-FIOCRUZ, Rio de Janeiro, Brazil) provided the animals, who were 7 weeks old when they arrived at the laboratory. All of the mice were kept in normal mouse plastic cages with stainless steel lids and bedding made of white Pinus wood shavings (5 mice per cage). Animal cages were kept in a controlled environment (22°C, about 70% relative humidity, and a 12-hour light/dark cycle with lights on at 8 a.m.) and were given free access to filtered tap water and commercially available pelleted food for rats and mice (Nuvital; Nuvilab, Curitiba, PR, Brazil).

DBA Intermediates

Concerns about ethics

The FIOCRUZ Ethics Committee on Animal Use accepted the experimental procedure (2012, P-84/10-7).

Treatment

Mice (n = 6) were given freshly produced PQ solutions in ultrapure water (2mg PQ base•mL-1 = 3.551mg PQ diphosphate•mL-1). PQ concentrations are measured as PQ base per milliliter-1. PQ aqueous solutions were stored refrigerated (4°C) and light-protected until used within 24 hours of preparation. Oral gavage was used to give PQ diphosphate dissolved in water at dosages comparable to 20 mg PQ•kg-1. Cardiovascular puncture was used to obtain heparinized mouse blood samples 15, 30, 60, and 90 minutes after PQ administration. [DBA Intermediates]

Samples of plasma

Centrifugation at 12,000 rcf for 15 minutes separated plasma from heparinized whole blood. Plasma samples were immediately transferred to Eppendorf tubes after separation and stored at -20°C until analyses, which were completed within 7 days of PQ delivery.

DBA Intermediates

Procedure for extracting PQ

PP was utilised, followed by drug extraction from plasma, due to its simplicity and inexpensive cost. The following PP conditions were empirically optimised and standardised. 50L of plasma was transferred to 1.5-mL Eppendorf® vials, which were then filled with 50 L of acetonitrile acidified with 2% acetic acid (w/v).

For 30 seconds, the tubes containing plasma and acetonitrile were vortexed carefully, avoiding any contact of the mixture with the tube tip. After that, each tube received 25L of an aqueous 12.5 percent zinc sulphate solution (w/v), which was vortexed for 30 seconds. The suspension was then allowed to stand for 30 minutes to allow the plasma protein to fully precipitate. After centrifuging the tubes at 12,000 rcf for 15 minutes, the supernatant was analysed using HPLC-DAD-UV. [DBA Intermediates]

chromatographic conditions and equipment

The Shimadzu Class-VP (liquid chromatographer attached to a Shimadzu UV detector with the diode array SPD M10A VP equipped with a SCL 10A VP controller, DGU14A degasser, 10ADVP LC binary pump, CTO 10ASVP oven, and SIL10AF autosampler) was used to perform the analyses.

Shimadzu Class VP® software, version 6.1, was used to analyse the chromatograms. As mobile phases, several combinations of acetonitrile, methanol, and ammonium acetate buffer were utilised. Before use, all buffer solutions were filtered via a Merck-Millipore 0.45-m pore PVDF filter. Silica-based C18 (250mm 4.6mm i.d. 5m, ODS Hypersil, Thermo, Waltham, MA, USA) and modified-silica cyanopropyl (250mm 4.6mm i.d. 5m, Supelcosil LC-CN; Supelco, St. Louis, MO, USA) HPLC columns were used. For all analyses, the injection volume was 20L.

DBA Intermediates

Validation

The newly developed method was validated using regulatory guidance documents such as the Brazilian Health Surveillance Agency’s (ANVISA) validation manual RE 899/2003, the National Institute of Metrology, Quality and Technology’s (Inmetro) guidance on the validation of analytical methods (DOQ-CGCRE-008/ 2007), ICH technical requirements for validation of analytical procedures (1995, 1996), and HPLC analysis manuals and handbooks. Selectivity, linearity, intra-day precision, inter-day precision, accuracy, recovery, robustness, and limits of detection and quantification were the metrics utilised to evaluate analytical performance.

Standard PQ solutions in ultrapure water were used to create calibration curves for nine concentrations (13, 30, 50, 100, 250, 500, 1,000, 1,500, and 2,000ng.mL-1). Three calibration curves were collected on three different days to assess linearity. A study using blank mouse plasma and mouse plasma spiked with a PQ solution of 250ng.mL-1 determined selectivity. [DBA Intermediates]

Homoscedasticity was determined by comparing experimental and estimated values obtained by fitting a linear model to calibration data. Five distinct PQ concentrations (13, 100, 250, 500, and 2,000ng.mL-1) were measured in duplicate three times over a single day to determine intra-day precision.

These five concentrations were tested in duplicate once a day on three different days to assess inter-day accuracy. The accuracy of calibration curves (13, 100, 250, 500, and 2,000ng.mL-1) was determined using the formula accuracy = (EAVR/TC) 100, where EAVR is the experimental average and TC is the theoretical concentration. For spikes of high, midrange, and low PQ concentrations, recovery was determined (250, 500, and 1,000ng.mL-1, respectively). [DBA Intermediates]

With seven chromatographic variables: buffer concentration, acetonitrile percentage in the mobile phase, flow rate, mobile phase pH, oven temperature, precipitation duration, and brand of commercially available acetonitrile, robustness was tested using Youden’s test.

With a PQ concentration of 500ng.mL-1, these seven parameters were mixed in eight random chromatographic trials (nominal condition + seven variants) (corresponding to an intermediate point along the standard analytical curve). Using the equations LOQ = DPa (standard deviation of intercept Y) 10/IC (curve slope) and LOD = DPa 3/IC, the limits of detection (LOD) and quantification (LOQ) were calculated from three calibration curves.

Analytical statistics

The information is presented as means and standard deviations. One-way ANOVA was used to examine differences between two means, followed by Bonferroni post-hoc testing. When p 0.05, differences were judged statistically significant. GRAPHPAD PRISM6® was used for statistical analysis.

DBA Intermediates

RESULTS

A new approach was developed based on laboratory analytical methodologies and previously published HPLC methods for antimalarial drug analysis. The performance of various acetonitrile or methanol combinations with acetate buffer as the mobile phase and RP-C18 silica or modified-silica cyanopropyl columns as the stationary phase was investigated.

Using a modified-silica cyanopropyl column (250mm 4.6mm i.d. 5m, Supelcosil LC-CN) and a 45:55 mixture of acetonitrile and 10mM ammonium acetate buffer (pH = 3.80) as the mobile phase, optimal HPLC conditions for the measurement of PQ in blood plasma were found. The use of organic mobile phase modifiers turned out to be unnecessary. [DBA Intermediates]

The flow rate was 1.0 mL.min-1, the temperature was 50 degrees Celsius, and the absorbance (UV) was measured at 264 nanometers. The proposed method for measuring PQ in blood plasma has a quick analysis time (t R = 5.80 0.20 min and a total analysis time of 7 min), strong signal symmetry (1.14), a high retention factor (k = 2.15), and low detection and quantification limitations.

Analyses of blank plasma samples and plasma samples spiked with PQ were used to determine the method’s selectivity and specificity. A, B, and C depict the PQ signal from the plasma extract at 5.94 min, blank plasma, and overlapping chromatograms, respectively. Around t R 5.9 min, no interference was identified for the PQ chromatographic window, demonstrating the method’s selectivity and specificity.

For 50-L plasma samples, a method for extracting PQ from the plasma was developed. Acetonitrile was added first, followed by zinc sulphate to achieve full PP (see Methods). The overall time required for complete PP and PQ extraction was around 45 minutes.

DBA Intermediates

PQ recoveries were 89.8 5.1 percent at 250ng.mL-1, 97.7 1.1 percent at 500ng.mL-1, and 100.4 4.6 percent at 1,000ng.mL-1 from plasma spiked with PQ diphosphate (stated as PQ base). The recovery of 80 percent or greater additional PQ at all tested concentrations20,23 revealed that the extraction procedure designed for small sample volumes (50L) was highly efficient.

The linearity, intra-day and inter-day precision, accuracy, recovery, robustness, and detection and quantification limitations of the HPLC-DAD-UV technique for the determination of PQ in plasma matrices developed in this work were all validated (see Methods). Excellent linearity was observed in the calibration curves generated in the concentration range of 13 to 2,000ng.mL-1 (r = 0.9997 0.0003; average and standard-deviation of three different calibration curves). [DBA Intermediates]

In the concentration range of 13 to 2,000ng.mL-1, the discrepancy between experimental and calculated values revealed that the distributions of values were homoscedastic. Concentration = [(absorbance) + 185.58 (36.14)]/103.29 (0.67) was the linear regression equation for PQ quantification in plasma samples. The calculated LOQ and LOD were 3.5 and 1.0ng.mL-1, respectively (see Methods).

Precision and accuracy are offered both intra-day and inter-day. Values for precision and accuracy were within acceptable limits (85-115 percent ). The method revealed the most variation in PQ content as a function of the mobile phase pH (13.7%) and, to a lesser extent, as a function of the acetonitrile brand (0.9 percent )

DBA Intermediates

DISCUSSION

HPLC-UV methods for PQ analysis in plasma that have previously been reported require large sample volumes (100-500L) and were developed and validated using human blood plasma. We developed a new extraction approach for PQ analysis in small amounts of plasma, which is required for investigations in small animals (such as DBA-2 mice), the most common experimental model of blood stage malaria. [DBA Intermediates]

DBA-2 mice are vulnerable to Plasmodium berghei (ANKA) infection and are commonly employed to research the physiopathology of severe and deadly malaria as well as to test new antimalarial drugs. Furthermore, the extraction technique is straightforward because it does not require solid phase extraction; the overall analysis time is brief (7 minutes); and the mobile phase consists of ultrapure water buffer and acetonitrile, with no organic modifiers required. The method is likely to be more cost-effective than traditional analytical methods for determining PQ in blood plasma.

Because a UV detector was utilised, the observed LOQ and LOD (3.5 and 1.0ng.mL-1, respectively) were extremely low. In fact, LOQs for PQ studies utilising HPLC-UV have been reported in the literature as high as 10ng.mL-1. The new method’s outstanding signal symmetry and linearity over a wide range of concentrations resulted in significantly lower LOQ and LOD estimations than similar HPLC methods for PQ previously described. [DBA Intermediates]

Variation in robustness varied from 0.9 percent (acetonitrile brand) to 13.7 percent (acetonitrile brand) (mobile phase pH). Nonetheless, fluctuation in any of the seven evaluated factors had no significant effect on the analytical assessment of PQ content (variation 15%), suggesting that the procedure is reasonably robust. In triple analyses of female DBA-2 blood plasma, the relative standard deviation was less than 5%, suggesting that drug plasma extraction and analysis are effective and repeatable.

It was possible to develop a new HPLC method for the quantification of PQ in small volumes of plasma that is suitable for kinetic studies of this compound in small rodents, including mouse models for the study of malaria, by combining a simple and relatively low-cost extraction procedure with a robust and validated analysis method. Selectivity, linearity, intra-day and inter-day precision, resilience, accuracy, and recovery are all strengths of this innovative approach. A quicker analysis time, the use of smaller amounts of plasma, and lower LOD and LOQs are all advantages of the analytical method over similar, current HPLC-UV procedures.

DBA Intermediates

How helpful is Chemical CAS No. ? →