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Development and validation of a HPLC-UV method for methadone hydrochloride quantification in a new oral solution with preservatives to be implemented in physicochemical stability studies

Development and validation of a HPLC-UV method for methadone hydrochloride quantification in a... Purpose: The Pharmacy Service of the Infanta Leonor University Hospital acquires, compounds, distributes and dis‑ penses more than 3000 L of methadone oral solution to Drug Addiction Patients Centers per year. Our purpose is to develop and validate an improved high performance liquid chromatography (HPLC) method to quantify methadone hydrochloride in a new oral solution with methylhydroxybenzoate (methylparaben) and propylhydroxybenzoate (propylparaben) to be implemented in physicochemical stability studies that allow to provide more information and even to increase the beyond‑use date. Methods: A HPLC‑Agilent 1100 equipment, comprising a quaternary pump and an ultraviolet diode‑array‑ detector (DAD) was used. An analytical method development and validation was completed. The curve was constructed from methadone working concentrations of 75–125% (7.5, 9.0, 10.0, 11.0 and 12.5 mg/mL) to assess the linear relationship between the concentration of the analyte and the obtained areas. Precision and accuracy were calculated. Detection and quantification limit (LD, LQ) were estimated using the EURACHEM method. Forced‑ degradation studies were also performed. Results: Chromatographic conditions were: flow rate 1.6 mL/min; mobile phase 55% acetonitrile and 45% sodium phosphate 25 mM (pH = 10); injection volume was 5 µL. The column was a Waters‑ XTerra RP18, maintained at 40 °C. DAD was λ = 254 nm. Retention times for methadone, methylparaben and propylparaben were 4.34, 0.70 and 0.88 min respectively. The method was linear (y = 284.3x − 97.8, r = 0.996). Instrumental precision was 0.33% for standards (n = 10); intra‑ assay precision 0.53% (n = 6) and inter‑assay precision 1.95% (n = 12). The relative standard deviation percentage for accuracy was 1.28%. The recovery percentage was 101.5 ± 1.5%. LQ and LD were 2.18 µg/mL and 2.0 µg/mL respec‑ tively. The most destabilizing conditions were oxidizing and alkaline. The chromatograms confirmed no interference with the methadone signal. *Correspondence: elenaalba.alvaro@salud.madrid.org Pharmacy Department, Infanta Leonor University Hospital, Av. Gran Vía del Este, 80, 28031 Madrid, Spain Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. ÁlvaroA ‑ lonso et al. BMC Chemistry (2022) 16:32 Page 2 of 10 Conclusions: The HPLC method has proved to be valid and reproducible for methadone quantification in a new oral solution with methylparaben and propylparaben. This assay is a rapid, simple and reliable technique that can be used in daily analysis and physicochemical stability studies. Keywords: Methadone hydrochloride, Pharmaceutical solutions, Drug compounding, High performance liquid chromatography, Analysis, Analytical chemistry method Introduction following disadvantages: lenghthy and wasteful use of In the Autonomous Community of Madrid, Resolution solvent analysis (gradient elution over 10–20 min), a tedi- 189/2018 [1] was implemented in March 2018, which ous extraction procedure (not suited for routine analysis) tasked the Hospital Pharmacy Service (HPS) of the or the use of a fluorescent ion-pairing agent [6–10]. Infanta Leonor University Hospital with supplying meth- It should be note that the methadone analyzed with adone to the 27 Centres for the Comprehensive Care of these methods was either found in concentrations dif- Drug Addiction Patients of the Madrid Health Care Ser- ferent from 10  mg/ml, or they were preparations for vice where methadone maintenance programs (MMP) for intravenous administration, or they were compounded opiate addictions are implemented. The aim of this Reso - with other vehicles than water (sodium chloride [11, lution was to centralize the acquisition, preparation, dis- 12], various drinks [13], syrups, suspension and sugar- tribution, and dispensing of methadone by the HPS. This free vehicles [14]) and none of the pharmaceuticals ana- initiative represented a first step in changing the pharma - lyzed for oral administration contained exactly the same cotherapeutic health care model for the treatment of the preservatives (only methylparaben) [9] used in our new patients in the program. To date, between 3000 and 5000 formulation [15], so they really were not suitable for us. patients are prescribed methadone as an opiate substitute Furthermore, these methods use columns that are no for the treatment of heroin-related addiction disorders. longer used, such as µBondapak or Radpak-Novapak, a The methadone solution prepared and supplied by type A silica-based column with a lot of silanolic activity the HPS to MMP patients is described in the Spanish and therefore a high possibility of deformation tailing. National Formulary [2] and is formulated with metha- Other studies performed since 2000 were focused on done hydrochloride in the raw material form and purified the development of new techniques to determine meth- water. A beyond-use date (BUD) of 30 days and refriger- adone in biological samples such as plasma, saliva and ated storage have been established. For this reason, and urine [16, 17] and even in wastewater [18–20], or by due to the large volume of methadone solution to be dis- HPLC-Ion-Trap Mass Spectrometry [21], electrochemi- pensed (around 3500 L per year), one of the future chal- cal detection [22] or other extraction techniques. These lenges [3] consists in carrying out a physicochemical methods, despite being more precise and having been and microbiological stability study in order to confirm developed in more complex matrices than our solution, and even increase its BUD as well as the development of are more complex and expensive techniques. Therefore, improvements to the formulation of the methadone solu- there would not be suitable for our work. Methadone tion by adding preservatives. Thus, a new compounding stability studies have also been described, but the meth- of methadone hydrocloride in oral solution was designed adone determination technique is either the USP one, and validated. Its composition included methylhyparaben those previously shown in the literature, GC [23] or spec- and propylparaben as preservatives. The final concentra - trophotometry [14]. tion was also 10 mg/mL. New analytical methods are always sought in order to It was necessary to determine and quantify the meth- obtain more and better information, with less consump- adone hydrochloride in the new oral solution without tion or contamination, in less time and with less effort. interference from the preservatives. In addition, analytical method development and valida- Methadone is an extensively studied drug and accord- tion procedures are vital in the discovery and develop- ing to the Spanish Pharmacopoeia [4], the technique of ment of drugs and pharmaceuticals to ensure the method choice is Gas Chromatography (GC). However, in the performance. United States Pharmacopoeia [5] (USP), quantitation The aim of this study was to develop and validate a sim - methods for methadone in pharmaceutical preparations ple, rapid and reproducible analytical method to quantify are based on acid titration, UV determination, GC, and methadone hydrochloride in a new oral solution with High Performance Liquid Chromatography (HPLC). methylparaben and propylparaben to be implemented Some of these methods present, as do HPLC methods in physicochemical stability studies. The future goal is described in the literature for methadone analysis, the to increase the BUD of methadone hydrochloride oral Á lvaroA ‑ lonso et al. BMC Chemistry (2022) 16:32 Page 3 of 10 solution to improve organizational aspects of the work- Linearity flow of HPS and increase adaptability to the individual A standard solution (100  mL) of methadone 50  mg/mL dispensing needs of patients. It will also be necessary to was prepared from which, by means of serial dilutions, carry out a physicochemical and microbiological stabil- a total of 20 calibration standards were obtained for ity study in different conservation environments of the the linearity test (four replicates for each concentration new formulation proposed in this work to provide more level). The curve was constructed from methadone work - information. ing concentrations of 75–125% (7.5, 9.0, 10.0, 11.0 and 12.5  mg/mL) to assess the linear relationship between the concentration of the analyte and the obtained areas. Methods Once the regression equation was obtained [26], an anal- Reagents, reference standards and materials ysis of variance (ANOVA) was performed. It has been used methadone hydrochloride purchased from Laboratorios Dr. Esteve S.A. (Barcelona, Spain). Precision As preservatives, methylparaben and propylparaben, Instrumental precision (repeatibility), intra-assay preci- acquired from Fagron Iberica (Terrassa, Spain) were sion and inter-assay precision (intermediate precision) used. Purified water was obtained from Grifols labora - were measured. For instrumental precision, a standard tory (Barcelona, Spain). All of them were of Pharmaco- solution (10 mL) of methadone 10 mg/mL was prepared poeia grade. by the same analyst on a single day and consecutively In the mobile phase we used acetonitrile HPLC grade, analyzed ten times to check the repeatability of the purchased from VWR Prolabo Chemicals (Fontenay- method and to assess the dispersion degree among the Sous-Bois, France). Phosphoric acid, sodium hydroxide series of measurements obtained. For intra-assay pre- (> 99%), hydrochloric acid and hydrogen peroxide were cision, six standards of methadone solution 10  mg/mL supplied from Panreac (Barcelona, Spain) and Milli-Q were prepared and analyzed. Inter-assay precision was water. All reagents and solvents were of analytical grade. also performed in another six standards of 10  mL of methadone solution 10  mg/mL which were prepared on a second day by different analysts, obtaining a total of 12 Equipment samples. HPLC analyses were performed on a qualified and cali - brated chromatography system, Agilent-Technologies Accuracy 1100 series (Madrid, Spain) comprising a quaternary The accuracy of the method was determined through gradient pump, an ultraviolet photodiode-array detec- spike recovery of the methadone solution with a pre- tor (UV-DAD), a 100-vial programmable autosampler, a servative matrix, diluted within the range used for final column oven compartment, an automatic injector and a sample measurements, and within the range of the software controller. corresponding calibration curves. Afterwards, three 10  mL replicates of three concentration levels 7.5, 10 and 12.5  mg/mL were prepared by serial dilutions from Chromatographic conditions TM  100  mL of a 50  mg/ml stock solution. The recovery per - We have used a Waters-XTerra RP18 (3.5  μm;4.6 centage and relative standard deviation percentage × 100 mm) column. The column temperature was main - (%RSD) were calculated. The maximum aceptable levels tained at 40  °C. The mobile phase consisted of acetoni - were 10%. trile as the organic phase (55%) and sodium phosphate 25 mM (adjusted to pH = 10) as the aqueous phase (45%). The flow rate was 1.6 mL/min. The injection volume was Detection and quantification limit 5  µL for each chromatographic analysis. The UV-DAD Detection limit (LD) and quantification limit (LQ) in case was set at λ = 254 nm. of instrumental method, can be estimated using various equations. However, in this assay, it was decided to use an experimental method, following EURACHEM recom- Validation of the HPLC method mendations [27] which consisted of preparing a series The methods and their acceptance criteria were estab - of samples with decreasing amounts of analyte and ana- lished on the basis of the International Conference on lyzing each of them six consecutive times, representing Harmonization (ICH) guidelines Q2 (R1) [25]. %RSD of the precision against the concentration of each sample. For this purpose, we prepared a battery of serial dilutions from a stock solution of methadone 20  mg/ ÁlvaroA ‑ lonso et al. BMC Chemistry (2022) 16:32 Page 4 of 10 mL. The concentrations were 2, 0.2, 0.02, 0.002 and with the parabens. In our analysis conditions, methadone 0.0002 mg/mL. Six replicates of each concentration were appears at a retention time of 4.34  min (which is 50% prepared, from which the area and retention times were less than in the USP monograph) [5] and methylparaben obtained. Normally, a precision criterion of %RSD of and propylparaben at 0.70 and 0.88 min respectively. The 10% is set at the LQ although up to 20% can be accepted, chromatograms obtained are shown in Figs. 1, 2 and 3. depending on the characteristics of the method. Both were also expressed as a percentage of the theoretical concentration. Linearity ANOVA analysis confirmed the linearity of the Forced‑degradation studies method in the range tested. The equation obtained was The methadone hydrochloride 10  mg/ml oral solution y = 284.3x − 97.8 being the correlation coefficient (r) was subjected to the following denaturing conditions to 0.996 and determination coefficient (r ) of 0.991. The determine the capacity of the HPLC method in order to results indicate that there is no significant statistical dis - detect any possible degradation products produced dur- persion between the results of the replicates of the differ - ing storage: in acid (0.1 M HCl at 25 °C), in base (0.1 M ent concentrations, with a correlation and determination NaOH at 25 °C) and in oxidation (3% H O at 25 °C). For coefficient greater than 0.99, corroborating compliance 2 2 this, 0.1 mL of methadone 10 mg/mL was diluted in 1 mL with the linearity method. of each denaturing reagent and they were kept in contact for 1  h until analysis. Then, following the same chroma - tographic conditions, elution cycles of 60 min were pro- Precision and accuracy grammed, and the test was carried out eight times for The results of instrumental precision, intra-assay and each stress condition. Peak purity was also calculated inter-assay precision and accuracy are shown in Tables 1, using the Agilent-ChemStation software tool based on 2. The percentage of recovery for all samples fulfilled the the similarity factor. requirements of the compounding stability studies (90– 110%) [28]. The small percentage of difference between Results the nominal and found concentration of the standards The HPLC method has demonstrated that there is no showed that the assay is sufficently accurate for their substance that interferes in the analysis of the different application. In addition, the %RSD value was below 10% formulations of methadone. It has been verified that, at at all concentrations, indicating that the assay method the analysis wavelength, 254 nm, there is no interference was reproducible across days. Fig. 1 Chromatogram of methadone hydrochloride 10 mg/mL with preservatives Á lvaroA ‑ lonso et al. BMC Chemistry (2022) 16:32 Page 5 of 10 Fig. 2 Chromatogram of methadone hydrochloride 10 mg/mL without preservatives Fig. 3 Chromatogram of blank Detection and Quantification Limit Interpolating in the obtained curve a %RSD = 10%, After representing %RSD versus concentration, a poten- we calculated that LQ was 2.18  µg/mL. The LD is the tial adjustment was observed. The equation obtained was value capable of detecting the analyte. However, in the −0.37 y = 0.0051x . We observed that as the concentration 0.0002  mg/mL concentration, four of the six samples decreases, %RSD increases due to the difficulty of detect - were not detected, so the LD was considered to be the ing methadone. For 0.002 mg/mL, %RSD was 11.3%. For previous concentration value in which methadone was 0.0002 mg/mL, there were four samples with no signal, so detected. Therefore, LD was 2.0 µg/mL. Expressing these we were unable to obtain the area value. values as a percentage of the theoretical concentration, ÁlvaroA ‑ lonso et al. BMC Chemistry (2022) 16:32 Page 6 of 10 Table 1 Accuracy results Sample Concentration (mg/mL) Area Recovery percentaje Intra‑assay accuracy 1 7.5 2156.23 101.77% 2 2156.13 101.76% 3 2150.20 101.50% 4 10 2834.91 99.30% 5 2792.10 97.85% 6 2838.16 99.41% 7 2843.81 99.60% 8 2852.00 99.88% 9 2864.73 100.31% 10 12.5 3526.82 98.18% 11 3599.04 100.14% 12 3606.48 100.34% Median 100.00% %RSD 1.27% Second‑assay accuracy 1 7.5 2044.37 96.72% 2 2034.18 96.26% 3 2015.79 95.43% 4 10 2725.24 95.59% 5 2719.33 95.39% 6 2714.90 95.24% 7 2718.15 95.35% 8 2713.72 95.20% 9 2733.81 95.88% 10 12.5 3520.51 98.01% 11 3527.89 98.21% 12 3532.32 98.33% Median 96.30% %RSD 1.22% Inter‑assay accuracy Median 98.15% %RSD 1.24% Table 2 Precision and accuracy results Instrumental precision (%CV) Intra‑assay precision Inter‑assay precision Precision (%CV) Accuracy (%) RSD (%) Precision (%CV) Accuracy (%) RSD (%) 0.33 (n=10) 0.5 (n=6) 100 1.3 2.0 (n=12) 98.15 1.2 LQ and LD were 0.022% and 0.02%. This means that the products from our drug peak of interest (in all situations, method is capable of detecting up to 0.02% and quantify- the methadone peak continued to be obtained at minute ing 0.022% of methadone contained in a 10  mg/mL oral four). The methadone peak purity was 999,830 over 1000 solution (the objective of our method was to quantify (1000 indicates identical spectra and values > 995 indicate methadone at around 100% concentration). that the spectra are very similar). The most destabilizing conditions were oxidizing Forced‑degradation studies and alkaline. In basic medium, turbidity was observed The results show that the method was stability-indi - practically instantaneously, indicating insolubility of cating, with complete separation of the degradation the methadone under this condition. In an acidic and Á lvaroA ‑ lonso et al. BMC Chemistry (2022) 16:32 Page 7 of 10 oxidative environment, some degradation of metha- Discussion done was observed due to the fact that we obtained A full method validation should be performed for any recovery percentages of 88%. Figures  4, 5, 6 show the analytical method whether new or based upon literature chromatograms obtained which confirmed that none of [29] as this ensures that the method developed is repro- the peaks found interfered with the methadone signal ducible, stable, sensitive, robust, suitable and reliable for and that no degradation products appear after 60  min. its application in pharmaceutical analysis. This means that the method is capable of quantifying The ICH recommends evaluating linearity in the range methadone separated from degradation products. 80–120% [25]. In our case, a margin of 75–125% was cho- sen and the method demonstrated good linearity over Fig. 4 Chromatogram in base condition Fig. 5 Chromatogram in acid condition ÁlvaroA ‑ lonso et al. BMC Chemistry (2022) 16:32 Page 8 of 10 Fig. 6 Chromatogram in oxidizing condition the range assayed. The method was repeatable. Intra- at the value of 0.02%, so the method not only quantifies and inter-assay precision %RSD were < 10% indicating perfectly in the limit of 90–110% indicated by the phar- that the assay method was reproducible across days. The macopoeias, but also is capable of quantifying impurities accuracy of an analytical procedure expresses the close- or degradation products that could be present in 0.02% ness of agreement between the value which is accepted of the final sample analyzed, in our case, the new oral either as a conventional true value or an accepted refer- solution. ence value and the value found [25]. The small percentage The ability of the assay to detect methadone decompo - of difference between the nominal and found concentra - sition was demostrated by stressing a methadone sam- tion of the standards demonstrated that the assay is accu- ple in forced-degradation studies which showed that rate enough for its application. The mean recovery values the most destabilizing stress conditions were the oxidiz- obtained were 100.0% and 98.5% respectively. Higher ing and alkaline conditions. The peaks obtained did not recovery indicates an efficient extraction procedure and interfere with methadone. Therefore, we can infer that higher sensitivity and accuracy of the analytical method. under pH conditions according to the specifications Following the specifications for methadone hydrochlo - (< 6.5), the pH variation within said limits will not pro- ride in oral solution described in the USP, Spanish Phar- duce any product that interferes with methadone quanti- macopoeia and Spanish National Formulary [2, 4, 5], it fication. Thus, it has been demonstrated that our method contains no less than 90.0% and no more than 110.0% is suitable for the detection and quantification of metha - of methadone hydrochloride. Consequently, one of the done hydrochloride in the presence of degradation prod- objectives of our method was to quantify methadone ucts. It is important to highlight that methadone solution near 100% concentration. as final pharmaceutical product will never occur in these When the method is defined as a content assessment extreme conditions, so it is unncessary to characterize analysis, where we will always work in ranges distant and quantify the peaks found in the chromatograms. from the minimum detectable or quantifiable quantity On the other hand, to understand the chromatographic using the equipment, it is not necessary to determine LQ conditions chosen for this study, it is first necessary to and LD. However, they were calculated, on the one hand study the characteristics of the methods described in the to demonstrate this situation, and on the other hand, to literature, and secondly, to know the methadone hydro- have a more complete validation because it allows a bet- chloride behavior in order to improve the conditions ter understanding of the analytical method, and knowing described. In this sense, at the beginning of the method the minimum analyte amounts that can be quantified, design, we studied methadone characteristics, and based can be useful for other applications. The method has on this, we tested different conditions to choose the more been shown to be capable of detecting and quantifying suitable ones. Á lvaroA ‑ lonso et al. BMC Chemistry (2022) 16:32 Page 9 of 10 The methadone pKa is 8.3, so a high pH environment Conclusions was needed in which we make sure that methadone is The HPLC method reported in this study is a rapid, sim - totally deprotonated. It was necessary to choose a suit- ple, reliable and economical technique analytically vali- able column and a mobile phase to work at a high pH dated and has allowed for the efficient quantification of values. For this reason, the column chosen was the methadone hydrochloride in a new oral solution with TM  Waters-XTerra RP18, which in addition to being methylparaben and propylparaben as preservatives and a resistant (it allows working at pH up to 12), allowed us concentration and composition not previously analyzed to avoid the tailing factor, obtain short retention times in the literature. This procedure is a new and an improved and achieve a good resolution of the methadone peak, method in comparison to those described in the USP and in addition to being sufficiently effective in avoiding literature. It has great recovery and the advantage that possible interference between methylparaben, propyl- the ion-pairing technique was not required, thus saving paraben and methadone (Fig.  1). The XTerraRP (First- time and money, which are key aspects in these times. Generation-Hybrid-Filler) columns, combine the best Furthemore, the HPLC method reported can be used in properties of silica and polymer bonded phases with the daily analysis of the methadone solution batches pre- patented hybrid-particle-technology, which replaces pared in the HPS and also to perform physicochemical one in three silanols with a methyl group. The result stability studies in different conservation environments is a mechanically strong particle that can be used for over a period of time in order to increase the BUD. high pH separations; as a consequence the charge and Acknowledgements peak shapes of the basic compounds are improved. We would like to acknowledge the entire CEMBIO department of the San Our wavelength is the one collected inside the UV- Pablo Ceu University Faculty of Pharmacy for allowing us to use their material, facilities and equipment to carry out this work. We also want to thank the Sub‑ DAD and in USP method, where comparing with directorate of Pharmacy and Health Products and the Subdirection of Addic‑ 274  nm, a higher absorption was observed. Regarding tions of the Ministry of Health of the Autonomous Community of Madrid for the mobile phase, different percentages were tested the collaboration and support always received. All these entities have helped develop this Project and bring it to fruition. For their unqualified and total until 45–55% was reached, where the peaks were per- support, we extend special thanks to the Direction of the Infanta Leonor fectly separated and resolved in < 5 min. University Hospital, the Pharmacy Service, and other Central Services of the The evaluation of robustness was considered not Infanta Leonor University Hospital. We would also like to thank Brian Crilly Montagne, María Chuecos Lozano and Francisco Javier Rupérez Pascualena for necessary. Variations in the flow rate or temperature the editorial help in the preparation of this paper. will not put the resolution between paraben and meth- adone at risk, as the resolution between them is close Author contributions A‑AEA: Conceptualization, Formal analysis, Investigation, Methodology, to 20. Nevertheless, the pH of the mobile phase is a Writing‑ Original draft preparation. LMP: Data curation, Formal analysis, Meth‑ critical parameter due to the ionization of methadone odology, Writing‑Reviewing and Editing. E‑RI: Supervision; Writing‑Reviewing and the stability of the stationary phase. According to and Editing. A‑RA: Supervision; Writing‑Reviewing and Editing. All authors read and approved the final manuscript. the pKa of methadone and the column manufacturer´s directions, pH must be adjusted between 9.3 and 11. Funding Below 9.3 methadone peak will show lower retention This research did not receive any specific grant from funding agencies in the public, commercial, or not‑for ‑profit sectors. time and tailing, and above 11 the shelf life of the col- umn is diminished. Availability of data and materials In this study we needed an analytical method to The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. quantify methadone hydrochloride in a new oral solu- tion with preservatives, not previously analyzed in the Declarations literature. Comparing our results with those found in other HPLC methods developed in different metha- Ethics approval and consent to participate done formulations, we can affirm that our method is This study does not involve human participants. This study does not involve animal subjects. efficient, rapid, simple, capable of quantifying metha- done without interference from preservatives and Consent for publication better than the methods already described whose dis- This manuscript does not contain any individual person’s data in any form. advantages have been highlighted. Competing interests The method reported in this assay can also be used The authors declare that they have no competing interests. to carry out physicochemical stability studies in which Author details the possible methadone degradation can be detected Pharmacy Department, Infanta Leonor University Hospital, Av. Gran Vía del in different conservation environments over a period 2 Este, 80, 28031 Madrid, Spain. Center for Metabolomics and Bioanalysis of time. (CEMBIO), Facultad de Farmacia, Universidad CEU San Pablo, CEU Universities, Urbanización Montepríncipe, 28660 Boadilla del Monte, Spain. F acultad de ÁlvaroA ‑ lonso et al. 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Eurachem, Guidance Document No. WGD 2, Accreditation for Chemi‑ of methadone, phencyclidine, and metabolites by postcolumn ion‑pair cal Laboratories: Guidance on the Interpretation of the EN45000 Series extraction and on‑line fluorescent detection of the counterion with of Standards and ISO/IEC, Guide 25, 1993. https:// www. eurac hem. org/ applications. J Pharm Sci. 1983;72:630–5. https:// doi. org/ 10. 1002/ jps. images/ stori es/ Guides/ pdf/ Eurac hem_ CITAC_ QAC_ 2016_ EN. pdf 26007 20613. 28. European Medicines Agency. Guideline on Stability Testing: Stability 9. Adams P, Haines‑nutt R. High‑performance liquid‑ chromatographic Testing of Existing Active Substances and Related Finished Products. analysis of methadone hydrochloride in pharmaceuticals. 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Friciu MM, Alarie H, Beauchemin M, et al. Stability of methadone hydro‑ chloride for injection in saline solution. Can J Hosp Pharm. 2020;73:141–4. Publisher’s Note 13. Lauriault G, Lebelle M, Lodge B, et al. Stability of methadone in 4 vehicles Springer Nature remains neutral with regard to jurisdictional claims in pub‑ for oral‑administration. Am J Hosp Pharm. 1991;48:1252–6. lished maps and institutional affiliations. 14. Provenza N, Calpena AC, Mallandrich M, et al. Design of pediatric oral formulations with a low proportion of methadone or phenobarbital for the treatment of neonatal abstinence syndrome. Pharm Dev Technol. 2016;21:755–62. https:// doi. org/ 10. 3109/ 10837 450. 2015. 10557 65. 15. Ching MS, Stead ChK, Shilson AD. Stability of methadone mixture with methyl hydroxybenzoate as a preservative. Aust J Hosp Pharm. 1989;19:159–61. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : 16. Miguez‑Diez E, Pitarch‑Sierra A, Modamio P, et al. HPLC‑UV method devel‑ opment and validation for quantitative determination of methadone in fast, convenient online submission human plasma. Int J Clin Phar. 2013;35:1007–1007. thorough peer review by experienced researchers in your field 17. George R, Lobb M, Haywood A, et al. Quantitative determination of the enantiomers of methadone in human plasma and saliva by chiral column rapid publication on acceptance chromatography coupled with mass spectrometric detection. Talanta. support for research data, including large and complex data types 2016;149:142–8. https:// doi. org/ 10. 1016/j. talan ta. 2015. 11. 044. • gold Open Access which fosters wider collaboration and increased citations 18. Castiglioni S, Zuccato E, Crisci E, et al. Identification and measurement maximum visibility for your research: over 100M website views per year of illicit drugs and their metabolites in urban wastewater by liquid chro‑ • matography−tandem mass spectrometry. Anal Chem. 2006;78:8421–9. https:// doi. org/ 10. 1021/ ac061 095b. At BMC, research is always in progress. 19. Baker DR, Kasprzyk‑Hordern B. Multi‑residue determination of the sorp ‑ Learn more biomedcentral.com/submissions tion of illicit drugs and pharmaceuticals to wastewater suspended par‑ ticulate matter using pressurised liquid extraction, solid phase extraction http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Chemistry Springer Journals

Development and validation of a HPLC-UV method for methadone hydrochloride quantification in a new oral solution with preservatives to be implemented in physicochemical stability studies

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10.1186/s13065-022-00827-9
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Abstract

Purpose: The Pharmacy Service of the Infanta Leonor University Hospital acquires, compounds, distributes and dis‑ penses more than 3000 L of methadone oral solution to Drug Addiction Patients Centers per year. Our purpose is to develop and validate an improved high performance liquid chromatography (HPLC) method to quantify methadone hydrochloride in a new oral solution with methylhydroxybenzoate (methylparaben) and propylhydroxybenzoate (propylparaben) to be implemented in physicochemical stability studies that allow to provide more information and even to increase the beyond‑use date. Methods: A HPLC‑Agilent 1100 equipment, comprising a quaternary pump and an ultraviolet diode‑array‑ detector (DAD) was used. An analytical method development and validation was completed. The curve was constructed from methadone working concentrations of 75–125% (7.5, 9.0, 10.0, 11.0 and 12.5 mg/mL) to assess the linear relationship between the concentration of the analyte and the obtained areas. Precision and accuracy were calculated. Detection and quantification limit (LD, LQ) were estimated using the EURACHEM method. Forced‑ degradation studies were also performed. Results: Chromatographic conditions were: flow rate 1.6 mL/min; mobile phase 55% acetonitrile and 45% sodium phosphate 25 mM (pH = 10); injection volume was 5 µL. The column was a Waters‑ XTerra RP18, maintained at 40 °C. DAD was λ = 254 nm. Retention times for methadone, methylparaben and propylparaben were 4.34, 0.70 and 0.88 min respectively. The method was linear (y = 284.3x − 97.8, r = 0.996). Instrumental precision was 0.33% for standards (n = 10); intra‑ assay precision 0.53% (n = 6) and inter‑assay precision 1.95% (n = 12). The relative standard deviation percentage for accuracy was 1.28%. The recovery percentage was 101.5 ± 1.5%. LQ and LD were 2.18 µg/mL and 2.0 µg/mL respec‑ tively. The most destabilizing conditions were oxidizing and alkaline. The chromatograms confirmed no interference with the methadone signal. *Correspondence: elenaalba.alvaro@salud.madrid.org Pharmacy Department, Infanta Leonor University Hospital, Av. Gran Vía del Este, 80, 28031 Madrid, Spain Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. ÁlvaroA ‑ lonso et al. BMC Chemistry (2022) 16:32 Page 2 of 10 Conclusions: The HPLC method has proved to be valid and reproducible for methadone quantification in a new oral solution with methylparaben and propylparaben. This assay is a rapid, simple and reliable technique that can be used in daily analysis and physicochemical stability studies. Keywords: Methadone hydrochloride, Pharmaceutical solutions, Drug compounding, High performance liquid chromatography, Analysis, Analytical chemistry method Introduction following disadvantages: lenghthy and wasteful use of In the Autonomous Community of Madrid, Resolution solvent analysis (gradient elution over 10–20 min), a tedi- 189/2018 [1] was implemented in March 2018, which ous extraction procedure (not suited for routine analysis) tasked the Hospital Pharmacy Service (HPS) of the or the use of a fluorescent ion-pairing agent [6–10]. Infanta Leonor University Hospital with supplying meth- It should be note that the methadone analyzed with adone to the 27 Centres for the Comprehensive Care of these methods was either found in concentrations dif- Drug Addiction Patients of the Madrid Health Care Ser- ferent from 10  mg/ml, or they were preparations for vice where methadone maintenance programs (MMP) for intravenous administration, or they were compounded opiate addictions are implemented. The aim of this Reso - with other vehicles than water (sodium chloride [11, lution was to centralize the acquisition, preparation, dis- 12], various drinks [13], syrups, suspension and sugar- tribution, and dispensing of methadone by the HPS. This free vehicles [14]) and none of the pharmaceuticals ana- initiative represented a first step in changing the pharma - lyzed for oral administration contained exactly the same cotherapeutic health care model for the treatment of the preservatives (only methylparaben) [9] used in our new patients in the program. To date, between 3000 and 5000 formulation [15], so they really were not suitable for us. patients are prescribed methadone as an opiate substitute Furthermore, these methods use columns that are no for the treatment of heroin-related addiction disorders. longer used, such as µBondapak or Radpak-Novapak, a The methadone solution prepared and supplied by type A silica-based column with a lot of silanolic activity the HPS to MMP patients is described in the Spanish and therefore a high possibility of deformation tailing. National Formulary [2] and is formulated with metha- Other studies performed since 2000 were focused on done hydrochloride in the raw material form and purified the development of new techniques to determine meth- water. A beyond-use date (BUD) of 30 days and refriger- adone in biological samples such as plasma, saliva and ated storage have been established. For this reason, and urine [16, 17] and even in wastewater [18–20], or by due to the large volume of methadone solution to be dis- HPLC-Ion-Trap Mass Spectrometry [21], electrochemi- pensed (around 3500 L per year), one of the future chal- cal detection [22] or other extraction techniques. These lenges [3] consists in carrying out a physicochemical methods, despite being more precise and having been and microbiological stability study in order to confirm developed in more complex matrices than our solution, and even increase its BUD as well as the development of are more complex and expensive techniques. Therefore, improvements to the formulation of the methadone solu- there would not be suitable for our work. Methadone tion by adding preservatives. Thus, a new compounding stability studies have also been described, but the meth- of methadone hydrocloride in oral solution was designed adone determination technique is either the USP one, and validated. Its composition included methylhyparaben those previously shown in the literature, GC [23] or spec- and propylparaben as preservatives. The final concentra - trophotometry [14]. tion was also 10 mg/mL. New analytical methods are always sought in order to It was necessary to determine and quantify the meth- obtain more and better information, with less consump- adone hydrochloride in the new oral solution without tion or contamination, in less time and with less effort. interference from the preservatives. In addition, analytical method development and valida- Methadone is an extensively studied drug and accord- tion procedures are vital in the discovery and develop- ing to the Spanish Pharmacopoeia [4], the technique of ment of drugs and pharmaceuticals to ensure the method choice is Gas Chromatography (GC). However, in the performance. United States Pharmacopoeia [5] (USP), quantitation The aim of this study was to develop and validate a sim - methods for methadone in pharmaceutical preparations ple, rapid and reproducible analytical method to quantify are based on acid titration, UV determination, GC, and methadone hydrochloride in a new oral solution with High Performance Liquid Chromatography (HPLC). methylparaben and propylparaben to be implemented Some of these methods present, as do HPLC methods in physicochemical stability studies. The future goal is described in the literature for methadone analysis, the to increase the BUD of methadone hydrochloride oral Á lvaroA ‑ lonso et al. BMC Chemistry (2022) 16:32 Page 3 of 10 solution to improve organizational aspects of the work- Linearity flow of HPS and increase adaptability to the individual A standard solution (100  mL) of methadone 50  mg/mL dispensing needs of patients. It will also be necessary to was prepared from which, by means of serial dilutions, carry out a physicochemical and microbiological stabil- a total of 20 calibration standards were obtained for ity study in different conservation environments of the the linearity test (four replicates for each concentration new formulation proposed in this work to provide more level). The curve was constructed from methadone work - information. ing concentrations of 75–125% (7.5, 9.0, 10.0, 11.0 and 12.5  mg/mL) to assess the linear relationship between the concentration of the analyte and the obtained areas. Methods Once the regression equation was obtained [26], an anal- Reagents, reference standards and materials ysis of variance (ANOVA) was performed. It has been used methadone hydrochloride purchased from Laboratorios Dr. Esteve S.A. (Barcelona, Spain). Precision As preservatives, methylparaben and propylparaben, Instrumental precision (repeatibility), intra-assay preci- acquired from Fagron Iberica (Terrassa, Spain) were sion and inter-assay precision (intermediate precision) used. Purified water was obtained from Grifols labora - were measured. For instrumental precision, a standard tory (Barcelona, Spain). All of them were of Pharmaco- solution (10 mL) of methadone 10 mg/mL was prepared poeia grade. by the same analyst on a single day and consecutively In the mobile phase we used acetonitrile HPLC grade, analyzed ten times to check the repeatability of the purchased from VWR Prolabo Chemicals (Fontenay- method and to assess the dispersion degree among the Sous-Bois, France). Phosphoric acid, sodium hydroxide series of measurements obtained. For intra-assay pre- (> 99%), hydrochloric acid and hydrogen peroxide were cision, six standards of methadone solution 10  mg/mL supplied from Panreac (Barcelona, Spain) and Milli-Q were prepared and analyzed. Inter-assay precision was water. All reagents and solvents were of analytical grade. also performed in another six standards of 10  mL of methadone solution 10  mg/mL which were prepared on a second day by different analysts, obtaining a total of 12 Equipment samples. HPLC analyses were performed on a qualified and cali - brated chromatography system, Agilent-Technologies Accuracy 1100 series (Madrid, Spain) comprising a quaternary The accuracy of the method was determined through gradient pump, an ultraviolet photodiode-array detec- spike recovery of the methadone solution with a pre- tor (UV-DAD), a 100-vial programmable autosampler, a servative matrix, diluted within the range used for final column oven compartment, an automatic injector and a sample measurements, and within the range of the software controller. corresponding calibration curves. Afterwards, three 10  mL replicates of three concentration levels 7.5, 10 and 12.5  mg/mL were prepared by serial dilutions from Chromatographic conditions TM  100  mL of a 50  mg/ml stock solution. The recovery per - We have used a Waters-XTerra RP18 (3.5  μm;4.6 centage and relative standard deviation percentage × 100 mm) column. The column temperature was main - (%RSD) were calculated. The maximum aceptable levels tained at 40  °C. The mobile phase consisted of acetoni - were 10%. trile as the organic phase (55%) and sodium phosphate 25 mM (adjusted to pH = 10) as the aqueous phase (45%). The flow rate was 1.6 mL/min. The injection volume was Detection and quantification limit 5  µL for each chromatographic analysis. The UV-DAD Detection limit (LD) and quantification limit (LQ) in case was set at λ = 254 nm. of instrumental method, can be estimated using various equations. However, in this assay, it was decided to use an experimental method, following EURACHEM recom- Validation of the HPLC method mendations [27] which consisted of preparing a series The methods and their acceptance criteria were estab - of samples with decreasing amounts of analyte and ana- lished on the basis of the International Conference on lyzing each of them six consecutive times, representing Harmonization (ICH) guidelines Q2 (R1) [25]. %RSD of the precision against the concentration of each sample. For this purpose, we prepared a battery of serial dilutions from a stock solution of methadone 20  mg/ ÁlvaroA ‑ lonso et al. BMC Chemistry (2022) 16:32 Page 4 of 10 mL. The concentrations were 2, 0.2, 0.02, 0.002 and with the parabens. In our analysis conditions, methadone 0.0002 mg/mL. Six replicates of each concentration were appears at a retention time of 4.34  min (which is 50% prepared, from which the area and retention times were less than in the USP monograph) [5] and methylparaben obtained. Normally, a precision criterion of %RSD of and propylparaben at 0.70 and 0.88 min respectively. The 10% is set at the LQ although up to 20% can be accepted, chromatograms obtained are shown in Figs. 1, 2 and 3. depending on the characteristics of the method. Both were also expressed as a percentage of the theoretical concentration. Linearity ANOVA analysis confirmed the linearity of the Forced‑degradation studies method in the range tested. The equation obtained was The methadone hydrochloride 10  mg/ml oral solution y = 284.3x − 97.8 being the correlation coefficient (r) was subjected to the following denaturing conditions to 0.996 and determination coefficient (r ) of 0.991. The determine the capacity of the HPLC method in order to results indicate that there is no significant statistical dis - detect any possible degradation products produced dur- persion between the results of the replicates of the differ - ing storage: in acid (0.1 M HCl at 25 °C), in base (0.1 M ent concentrations, with a correlation and determination NaOH at 25 °C) and in oxidation (3% H O at 25 °C). For coefficient greater than 0.99, corroborating compliance 2 2 this, 0.1 mL of methadone 10 mg/mL was diluted in 1 mL with the linearity method. of each denaturing reagent and they were kept in contact for 1  h until analysis. Then, following the same chroma - tographic conditions, elution cycles of 60 min were pro- Precision and accuracy grammed, and the test was carried out eight times for The results of instrumental precision, intra-assay and each stress condition. Peak purity was also calculated inter-assay precision and accuracy are shown in Tables 1, using the Agilent-ChemStation software tool based on 2. The percentage of recovery for all samples fulfilled the the similarity factor. requirements of the compounding stability studies (90– 110%) [28]. The small percentage of difference between Results the nominal and found concentration of the standards The HPLC method has demonstrated that there is no showed that the assay is sufficently accurate for their substance that interferes in the analysis of the different application. In addition, the %RSD value was below 10% formulations of methadone. It has been verified that, at at all concentrations, indicating that the assay method the analysis wavelength, 254 nm, there is no interference was reproducible across days. Fig. 1 Chromatogram of methadone hydrochloride 10 mg/mL with preservatives Á lvaroA ‑ lonso et al. BMC Chemistry (2022) 16:32 Page 5 of 10 Fig. 2 Chromatogram of methadone hydrochloride 10 mg/mL without preservatives Fig. 3 Chromatogram of blank Detection and Quantification Limit Interpolating in the obtained curve a %RSD = 10%, After representing %RSD versus concentration, a poten- we calculated that LQ was 2.18  µg/mL. The LD is the tial adjustment was observed. The equation obtained was value capable of detecting the analyte. However, in the −0.37 y = 0.0051x . We observed that as the concentration 0.0002  mg/mL concentration, four of the six samples decreases, %RSD increases due to the difficulty of detect - were not detected, so the LD was considered to be the ing methadone. For 0.002 mg/mL, %RSD was 11.3%. For previous concentration value in which methadone was 0.0002 mg/mL, there were four samples with no signal, so detected. Therefore, LD was 2.0 µg/mL. Expressing these we were unable to obtain the area value. values as a percentage of the theoretical concentration, ÁlvaroA ‑ lonso et al. BMC Chemistry (2022) 16:32 Page 6 of 10 Table 1 Accuracy results Sample Concentration (mg/mL) Area Recovery percentaje Intra‑assay accuracy 1 7.5 2156.23 101.77% 2 2156.13 101.76% 3 2150.20 101.50% 4 10 2834.91 99.30% 5 2792.10 97.85% 6 2838.16 99.41% 7 2843.81 99.60% 8 2852.00 99.88% 9 2864.73 100.31% 10 12.5 3526.82 98.18% 11 3599.04 100.14% 12 3606.48 100.34% Median 100.00% %RSD 1.27% Second‑assay accuracy 1 7.5 2044.37 96.72% 2 2034.18 96.26% 3 2015.79 95.43% 4 10 2725.24 95.59% 5 2719.33 95.39% 6 2714.90 95.24% 7 2718.15 95.35% 8 2713.72 95.20% 9 2733.81 95.88% 10 12.5 3520.51 98.01% 11 3527.89 98.21% 12 3532.32 98.33% Median 96.30% %RSD 1.22% Inter‑assay accuracy Median 98.15% %RSD 1.24% Table 2 Precision and accuracy results Instrumental precision (%CV) Intra‑assay precision Inter‑assay precision Precision (%CV) Accuracy (%) RSD (%) Precision (%CV) Accuracy (%) RSD (%) 0.33 (n=10) 0.5 (n=6) 100 1.3 2.0 (n=12) 98.15 1.2 LQ and LD were 0.022% and 0.02%. This means that the products from our drug peak of interest (in all situations, method is capable of detecting up to 0.02% and quantify- the methadone peak continued to be obtained at minute ing 0.022% of methadone contained in a 10  mg/mL oral four). The methadone peak purity was 999,830 over 1000 solution (the objective of our method was to quantify (1000 indicates identical spectra and values > 995 indicate methadone at around 100% concentration). that the spectra are very similar). The most destabilizing conditions were oxidizing Forced‑degradation studies and alkaline. In basic medium, turbidity was observed The results show that the method was stability-indi - practically instantaneously, indicating insolubility of cating, with complete separation of the degradation the methadone under this condition. In an acidic and Á lvaroA ‑ lonso et al. BMC Chemistry (2022) 16:32 Page 7 of 10 oxidative environment, some degradation of metha- Discussion done was observed due to the fact that we obtained A full method validation should be performed for any recovery percentages of 88%. Figures  4, 5, 6 show the analytical method whether new or based upon literature chromatograms obtained which confirmed that none of [29] as this ensures that the method developed is repro- the peaks found interfered with the methadone signal ducible, stable, sensitive, robust, suitable and reliable for and that no degradation products appear after 60  min. its application in pharmaceutical analysis. This means that the method is capable of quantifying The ICH recommends evaluating linearity in the range methadone separated from degradation products. 80–120% [25]. In our case, a margin of 75–125% was cho- sen and the method demonstrated good linearity over Fig. 4 Chromatogram in base condition Fig. 5 Chromatogram in acid condition ÁlvaroA ‑ lonso et al. BMC Chemistry (2022) 16:32 Page 8 of 10 Fig. 6 Chromatogram in oxidizing condition the range assayed. The method was repeatable. Intra- at the value of 0.02%, so the method not only quantifies and inter-assay precision %RSD were < 10% indicating perfectly in the limit of 90–110% indicated by the phar- that the assay method was reproducible across days. The macopoeias, but also is capable of quantifying impurities accuracy of an analytical procedure expresses the close- or degradation products that could be present in 0.02% ness of agreement between the value which is accepted of the final sample analyzed, in our case, the new oral either as a conventional true value or an accepted refer- solution. ence value and the value found [25]. The small percentage The ability of the assay to detect methadone decompo - of difference between the nominal and found concentra - sition was demostrated by stressing a methadone sam- tion of the standards demonstrated that the assay is accu- ple in forced-degradation studies which showed that rate enough for its application. The mean recovery values the most destabilizing stress conditions were the oxidiz- obtained were 100.0% and 98.5% respectively. Higher ing and alkaline conditions. The peaks obtained did not recovery indicates an efficient extraction procedure and interfere with methadone. Therefore, we can infer that higher sensitivity and accuracy of the analytical method. under pH conditions according to the specifications Following the specifications for methadone hydrochlo - (< 6.5), the pH variation within said limits will not pro- ride in oral solution described in the USP, Spanish Phar- duce any product that interferes with methadone quanti- macopoeia and Spanish National Formulary [2, 4, 5], it fication. Thus, it has been demonstrated that our method contains no less than 90.0% and no more than 110.0% is suitable for the detection and quantification of metha - of methadone hydrochloride. Consequently, one of the done hydrochloride in the presence of degradation prod- objectives of our method was to quantify methadone ucts. It is important to highlight that methadone solution near 100% concentration. as final pharmaceutical product will never occur in these When the method is defined as a content assessment extreme conditions, so it is unncessary to characterize analysis, where we will always work in ranges distant and quantify the peaks found in the chromatograms. from the minimum detectable or quantifiable quantity On the other hand, to understand the chromatographic using the equipment, it is not necessary to determine LQ conditions chosen for this study, it is first necessary to and LD. However, they were calculated, on the one hand study the characteristics of the methods described in the to demonstrate this situation, and on the other hand, to literature, and secondly, to know the methadone hydro- have a more complete validation because it allows a bet- chloride behavior in order to improve the conditions ter understanding of the analytical method, and knowing described. In this sense, at the beginning of the method the minimum analyte amounts that can be quantified, design, we studied methadone characteristics, and based can be useful for other applications. The method has on this, we tested different conditions to choose the more been shown to be capable of detecting and quantifying suitable ones. Á lvaroA ‑ lonso et al. BMC Chemistry (2022) 16:32 Page 9 of 10 The methadone pKa is 8.3, so a high pH environment Conclusions was needed in which we make sure that methadone is The HPLC method reported in this study is a rapid, sim - totally deprotonated. It was necessary to choose a suit- ple, reliable and economical technique analytically vali- able column and a mobile phase to work at a high pH dated and has allowed for the efficient quantification of values. For this reason, the column chosen was the methadone hydrochloride in a new oral solution with TM  Waters-XTerra RP18, which in addition to being methylparaben and propylparaben as preservatives and a resistant (it allows working at pH up to 12), allowed us concentration and composition not previously analyzed to avoid the tailing factor, obtain short retention times in the literature. This procedure is a new and an improved and achieve a good resolution of the methadone peak, method in comparison to those described in the USP and in addition to being sufficiently effective in avoiding literature. It has great recovery and the advantage that possible interference between methylparaben, propyl- the ion-pairing technique was not required, thus saving paraben and methadone (Fig.  1). The XTerraRP (First- time and money, which are key aspects in these times. Generation-Hybrid-Filler) columns, combine the best Furthemore, the HPLC method reported can be used in properties of silica and polymer bonded phases with the daily analysis of the methadone solution batches pre- patented hybrid-particle-technology, which replaces pared in the HPS and also to perform physicochemical one in three silanols with a methyl group. The result stability studies in different conservation environments is a mechanically strong particle that can be used for over a period of time in order to increase the BUD. high pH separations; as a consequence the charge and Acknowledgements peak shapes of the basic compounds are improved. We would like to acknowledge the entire CEMBIO department of the San Our wavelength is the one collected inside the UV- Pablo Ceu University Faculty of Pharmacy for allowing us to use their material, facilities and equipment to carry out this work. We also want to thank the Sub‑ DAD and in USP method, where comparing with directorate of Pharmacy and Health Products and the Subdirection of Addic‑ 274  nm, a higher absorption was observed. Regarding tions of the Ministry of Health of the Autonomous Community of Madrid for the mobile phase, different percentages were tested the collaboration and support always received. All these entities have helped develop this Project and bring it to fruition. For their unqualified and total until 45–55% was reached, where the peaks were per- support, we extend special thanks to the Direction of the Infanta Leonor fectly separated and resolved in < 5 min. University Hospital, the Pharmacy Service, and other Central Services of the The evaluation of robustness was considered not Infanta Leonor University Hospital. We would also like to thank Brian Crilly Montagne, María Chuecos Lozano and Francisco Javier Rupérez Pascualena for necessary. Variations in the flow rate or temperature the editorial help in the preparation of this paper. will not put the resolution between paraben and meth- adone at risk, as the resolution between them is close Author contributions A‑AEA: Conceptualization, Formal analysis, Investigation, Methodology, to 20. Nevertheless, the pH of the mobile phase is a Writing‑ Original draft preparation. LMP: Data curation, Formal analysis, Meth‑ critical parameter due to the ionization of methadone odology, Writing‑Reviewing and Editing. E‑RI: Supervision; Writing‑Reviewing and the stability of the stationary phase. According to and Editing. A‑RA: Supervision; Writing‑Reviewing and Editing. All authors read and approved the final manuscript. the pKa of methadone and the column manufacturer´s directions, pH must be adjusted between 9.3 and 11. Funding Below 9.3 methadone peak will show lower retention This research did not receive any specific grant from funding agencies in the public, commercial, or not‑for ‑profit sectors. time and tailing, and above 11 the shelf life of the col- umn is diminished. Availability of data and materials In this study we needed an analytical method to The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. quantify methadone hydrochloride in a new oral solu- tion with preservatives, not previously analyzed in the Declarations literature. Comparing our results with those found in other HPLC methods developed in different metha- Ethics approval and consent to participate done formulations, we can affirm that our method is This study does not involve human participants. This study does not involve animal subjects. efficient, rapid, simple, capable of quantifying metha- done without interference from preservatives and Consent for publication better than the methods already described whose dis- This manuscript does not contain any individual person’s data in any form. advantages have been highlighted. Competing interests The method reported in this assay can also be used The authors declare that they have no competing interests. to carry out physicochemical stability studies in which Author details the possible methadone degradation can be detected Pharmacy Department, Infanta Leonor University Hospital, Av. Gran Vía del in different conservation environments over a period 2 Este, 80, 28031 Madrid, Spain. Center for Metabolomics and Bioanalysis of time. (CEMBIO), Facultad de Farmacia, Universidad CEU San Pablo, CEU Universities, Urbanización Montepríncipe, 28660 Boadilla del Monte, Spain. F acultad de ÁlvaroA ‑ lonso et al. 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Miguez‑Diez E, Pitarch‑Sierra A, Modamio P, et al. HPLC‑UV method devel‑ opment and validation for quantitative determination of methadone in fast, convenient online submission human plasma. Int J Clin Phar. 2013;35:1007–1007. thorough peer review by experienced researchers in your field 17. George R, Lobb M, Haywood A, et al. Quantitative determination of the enantiomers of methadone in human plasma and saliva by chiral column rapid publication on acceptance chromatography coupled with mass spectrometric detection. Talanta. support for research data, including large and complex data types 2016;149:142–8. https:// doi. org/ 10. 1016/j. talan ta. 2015. 11. 044. • gold Open Access which fosters wider collaboration and increased citations 18. Castiglioni S, Zuccato E, Crisci E, et al. Identification and measurement maximum visibility for your research: over 100M website views per year of illicit drugs and their metabolites in urban wastewater by liquid chro‑ • matography−tandem mass spectrometry. Anal Chem. 2006;78:8421–9. https:// doi. org/ 10. 1021/ ac061 095b. At BMC, research is always in progress. 19. Baker DR, Kasprzyk‑Hordern B. Multi‑residue determination of the sorp ‑ Learn more biomedcentral.com/submissions tion of illicit drugs and pharmaceuticals to wastewater suspended par‑ ticulate matter using pressurised liquid extraction, solid phase extraction

Journal

BMC ChemistrySpringer Journals

Published: May 14, 2022

Keywords: Methadone hydrochloride; Pharmaceutical solutions; Drug compounding; High performance liquid chromatography; Analysis; Analytical chemistry method

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