Development and validation of UPLC-MS/MS method for 5-Fluorouracil quantification in dried blood spot.

5-Fluorouracil is an antimetabolite drug indicated for cancer treatment. Therapeutic drug monitoring of 5-Fluorouracil is necessary because 5-Fluorouracil has narrow therapeutic window and its concentration in blood is affected by individual conditions, like gene polymorphisms. Dried Blood Spot (DBS) is one of the biosampling methods used for therapeutic drug monitoring. Asides from reducing patients' discomfort, the use of DBS can increase 5-Fluorouracil stability by stopping the enzymes activity in blood. Therefore, this research developed a method to monitor 5-Fluorouracil levels in DBS using ultra-high performance liquid chromatography-tandem mass spectrometry. Sample preparation was carried out by extracting DBS using 2-Propanol: ethyl acetate (16:84). Reconstituted samples were analyzed using ultra high performance liquid chromatography equipped with Acquity® UPLC BEH C18 column (2.1 × 100 mm; 1.7 µm). The ionization process was carried out in negative electrospray ionization mode. Multiple Reaction Monitoring (MRM) values were set at m/z 128.97 > 41.82 for 5-Fluorouracil and 168.97 > 57.88 for propylthiouracil as the internal standard. Optimum analytical conditions were obtained with acetonitrile-ammonium acetate 1 mM (95:5) as mobile phase, flow rate of 0.15 mL/min, and column temperature of 40 °C. The lowest level of quantification obtained from this method was 0.1 µg/mL with a calibration curve range of 0.1 µg/mL-60 µg/mL. This method was proven to be valid according to the requirements set by the US Food and Drug Administration and the European Medicines Agency.

Development and validation of ivermectin quantification method in volumetric absorptive microsampling using liquid chromatography-tandem mass spectrometry.

Background: Ivermectin is a broad-spectrum anthelmintic used to control onchocerciasis from nematode parasites. As an anthelmintic, ivermectin is designed to have high levels in the gastrointestinal tract, so that the systemic intake is relatively low. Due to the very small concentration of ivermectin, a selective and sensitive approach is needed for the analysis of ivermectin in blood. Several methods have been developed using plasma and Dried Blood Spots, but there are still shortcomings due to hematocrit effects. Therefore, this study was conducted to establish a validated ivermectin analysis method with doramectin as the internal standard in using Ultra High-Performance Liquid Chromatography-Tandem Mass Spectrometry. Methods: Mass spectrometry equipped with triple quadrupole and positive electrospray ionization mode was used to conduct the analysis. For the biological matrix, whole blood was used by Volumetric Absorptive Microsampling and extracted using a protein precipitation technique with a combination of acetonitrile and methanol (1:1). VAMS has some advantages such as not being affected by hematocrit, requires a small and fixed volume of sample, also a more efficient sampling process. Results: The optimum conditions were achieved with an Acquity® UPLC BEH C18 column (1,7 µm; 2.1 × 100 mm); extracted-flow rate was 0,2 mL/min; mobile phase was 5 mM ammonium formate pH 3.00 and acetonitrile (10:90) with isocratic elution. Multiple Reaction Monitoring (MRM) detection by m/z values was 892.41 > 569.5 for ivermectin and 916,41 > 331,35 for doramectin. Conclusion: The method has been appropriately validated in compliance with the 2018 guidelines laid out by the US Food and Drug Administration. Resulting the minimum detection (LLOQ) was 1 ng/mL with a linear concentration range spanning from 1 to 150 ng/mL.

Doxorubicin, doxorubicinol, cardiotoxicity, breast cancer, volumetric absorptive microsampling, LC-MS/MS.

<b>Background and Objective:</b> Doxorubicin is an anticancer therapy belonging to the anthracycline class, which has clinical activity in breast cancer. Doxorubicin can cause cardiotoxic effects due to the formation of doxorubicinol as its main metabolite. The purpose of this study was to obtain the optimum sample preparation conditions for the analysis of doxorubicin in VAMS and as a form of therapeutic drug monitoring (TDM) in patients with cancer breasts. <b>Materials and Methods:</b> Analyze doxorubicin and doxorubicinol levels with Volumetric Absorptive Microsampling (VAMS) in patients' cancer breasts receiving doxorubicin in their therapeutic regimen. The sample was analyzed using Ultra Performance Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS). The method uses deep linear range concentrations of 8-200 ng/mL for doxorubicin and 3-100 ng/mL for doxorubicinol. <b>Results:</b> Multiple reaction monitoring (MRM) value set at m/z 544.22>396.9 for doxorubicin; m/z 546.22>398.9 for doxorubicinol and m/z 528.5>362.95 for daunorubicin. The LLOQ value obtained was 8 ng/mL for doxorubicin and 3 ng/mL for doxorubicinol with linearity of 0.9904 for doxorubicin and 0.9902 for doxorubicinol. Analysis results show doxorubicin levels were in the range of 9.47 ng/mL to 87.84 ng/mL and doxorubicinol range between 4.24 and 54.02 ng/mL. <b>Conclusion:</b> Dosage cumulative doxorubicin ranges between 47.93 and 346.09 mg/m²; with this, the risk of cardiomyopathy in the patients surveyed is under 4%, according to the literature.

Vernonia amygdalina Ethanol Extract Protects against Doxorubicin-Induced Cardiotoxicity via TGFß, Cytochrome c, and Apoptosis.

Doxorubicin (DOX) has been extensively utilized in cancer treatment. However, DOX administration has adverse effects, such as cardiac injury. This study intends to analyze the expression of TGF, cytochrome c, and apoptosis on the cardiac histology of rats induced with doxorubicin, since the prevalence of cardiotoxicity remains an unpreventable problem due to a lack of understanding of the mechanism underlying the cardiotoxicity result. Vernonia amygdalina ethanol extract (VAEE) was produced by soaking dried Vernonia amygdalina leaves in ethanol. Rats were randomly divided into seven groups: K- (only given doxorubicin 15 mg/kgbw), KN (water saline), P100, P200, P400, P4600, and P800 (DOX 15 mg/kgbw + 100, 200, 400, 600, and 800 mg/kgbw extract); at the end of the study, rats were scarified, and blood was taken directly from the heart; the heart was then removed. TGF, cytochrome c, and apoptosis were stained using immunohistochemistry, whereas SOD, MDA, and GR concentration were evaluated using an ELISA kit. In conclusion, ethanol extract might protect the cardiotoxicity produced by doxorubicin by significantly reducing the expression of TGF, cytochrome c, and apoptosis in P600 and P800 compared to untreated control K- (p < 0.001). These findings suggest that Vernonia amygdalina may protect cardiac rats by reducing the apoptosis, TGF, and cytochrome c expression while not producing the doxorubicinol as doxorubicin metabolite. In the future, Vernonia amygdalina could be used as herbal preventive therapy for patient administered doxorubicin to reduce the incidence of cardiotoxicity.

Development and validation of method for analysis of favipiravir and remdesivir in volumetric absorptive microsampling with ultra high-performance liquid chromatography-tandem mass spectrophotometry.

Favipiravir and remdesivir are drugs to treat COVID-19. This study aims to find an optimum and validated method for simultaneous analysis of favipiravir and remdesivir in Volumetric Absorptive Microsampling (VAMS) by Ultra High-Performance Liquid Chromatography-Tandem Mass Spectrophotometry. The use of VAMS can be an advantage because the volume of blood is small and the sample preparation process is simple. Sample preparation was done by precipitation of protein using 500 µL of methanol. Analysis was carried out by ultra high-performance liquid chromatography-tandem mass spectrophotometry with ESI+ and MRM with m/z 157.9 > 112.92 for favipiravir, 603.09 > 200.005 for remdesivir, and at m/z 225.968 > 151.991 for acyclovir as the internal standard. The separation was carried out using an Acquity UPLC BEH C18 column (100 × 2.1 mm; 1.7 m), 0.2% formic acid-acetonitrile (50:50), flow rate was 0.15 mL/min, and column temperature was 50°C. The analytical method has been validated with the requirements issued by the Food and Drug Administration (2018) and European Medicine Agency (2011). The calibration range of favipiravir is 0.5-160 µg/mL and 0.002-8 µg/mL for remdesivir.

Combination of Dissolving Microneedles with Nanosuspension and Co-Grinding for Transdermal Delivery of Ketoprofen.

Ketoprofen is an anti-inflammatory agent that may cause gastric irritation if administered orally. Dissolving microneedles (DMN) can be a promising strategy to overcome this issue. However, ketoprofen has a low solubility; therefore, it is essential to enhance its solubility using certain methods, namely nanosuspension (NS) and co-grinding (CG). This research aimed to formulate DMN containing ketoprofen-loaded NS and CG. Ketoprofen NS was formulated with poly(vinyl alcohol) (PVA) at concentrations of 0.5%, 1%, and 2%. CG was prepared by grinding ketoprofen with PVA or poly(vinyl pyrrolidone) (PVP) at different drug-polymer ratios. The manufactured ketoprofen-loaded NS and CG were evaluated in terms of their dissolution profile. The most promising formulation from each system was then formulated into microneedles (MNs). The fabricated MNs were assessed in terms of their physical and chemical properties. An in vitro permeation study using Franz diffusion cells was also carried out. The most promising MN-NS and MN-CG formulations were F4-MN-NS (PVA 5%-PVP 10%), F5-MN-NS (PVA 5%-PVP 15%), F8-MN-CG (PVA 5%-PVP 15%), and F11-MN-CG (PVA 7.5%-PVP 15%), respectively. The cumulative amounts of drug permeated after 24 h for F5-MN-NS and F11-MN-CG were 3.88 ± 0.46 µg and 8.73 ± 1.40 µg, respectively. In conclusion, the combination of DMN with nanosuspension or a co-grinding system may be a promising strategy for delivering ketoprofen transdermally.

Enhancing Intradermal Delivery of Lidocaine by Dissolving Microneedles: Comparison between Hyaluronic Acid and Poly(Vinyl Pyrrolidone) Backbone Polymers.

Lidocaine hydrochloride (LiH), an amide-type local anesthetic agent, is commonly used in dermatological procedures. LiH is categorized as a BCS (biopharmaceutics classification system) class III group, which has high solubility and poor permeability. It should be noted that, in this context, LiH is intended as a local anesthetic, so the level of LiH in systemic circulation should be minimized to avoid toxicity and unwanted side effects such as hypotension and bradycardia. This study aimed to formulate and evaluate LiH-loaded dissolving microneedles (DMNs) with different polymer bases. Moreover, an in vitro permeation study using Franz diffusion cells and in vivo study were also performed. LiH-loaded DMNs were prepared using polymer groups of poly(vinyl pyrrolidone) (PVP-K30) and hyaluronic acid (HA). DMNs were created using the micro-molding method with centrifugation. The formulations selected based on the evaluation were F3 (HA 10%) and F5 (PVP-K30 25%). Based on the in vitro permeation study, the amount of drug permeated and deposited in the skin at F3 (HA 10%) was 247.1 ± 41.85 and 98.35 ± 12.86 µg, respectively. On the other hand, the amount of drug permeated and deposited in the skin at F5 (PVP-K30 25%) was 277.7 ± 55.88 and 59.46 ± 9.25 µg, respectively. Our in vivo drug-permeation study showed that only one rat from the PVP-K30 polymer group-with a concentration of 150.32 ng/mL-was detected on rat plasma. Therefore, LiH can be formulated into a DMN and can be deposited in the skin with a safe concentration of the drug permeating into systemic circulation.

HPLC-DAD quantification of favipiravir in whole blood after extraction from volumetric absorptive microsampling devices.

Favipiravir is a prodrug of T-1105 made by modifying the pyrazine group as a COVID-19 therapy. During the pandemic, a safe and comfortable biosampling technique is needed for the subject or patient. Volumetric Absorptive Microsampling (VAMS) is a biosampling technique with a small blood volume and minimum hematocrit effect. The aims of this study were to develop and validate an analytical method for quantifying favipiravir extracted from VAMS using High Performance Liquid Chromatography - Photodiode Array with remdesivir as an internal standard. Analysis of favipiravir was performed using a C18 column (Waters, Sunfire™ 5 µm; 250 × 4.6 mm), with injection volume of 50 µL, flow rate of 0.8 mL/min, column temperature 30 ℃, and wavelength 300 nm. The separation was conducted under gradient elution with mobile phase consists of acetonitrile-0.2 % formic acid-20 mM sodium dihydrogen phosphate pH 3.5 and run time 12 min. Sample preparation was carried out using a protein precipitation method with 500 µL of methanol as precipitating agent. Samples were mixed on vortex for 30 s, sonicated for 15 min, and centrifuged at 10,000 rpm for 10 min. Lower Limit of Quantification (LLOQ) obtained was 0.5 µg/mL and the calibration curve ranged from 0.5 to 160 µg/mL. Sensitivity, linearity, selectivity, carry-over, accuracy, precision, recovery, and stability were validated by the guideline from Food and Drug Administration 2018. The method developed has successfully met the full validation requirements by FDA 2018 with the LLOQ obtained was 0.5 µg /mL.

Analysis of N7-(2-carbamoyl-2-hydroxyethyl)guanine in dried blood spot after food exposure by Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry.

N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-CAG) is a DNA adduct formed by glycidamide, which is the metabolite of acrylamide. Acrylamide can be found in foods containing reducing sugars and asparagine that are heated at high temperatures. Analysis of N7-CAG was performed in Dried Blood Spot (DBS) samples from 25 subjects of group test who consumed a lot of acrylamide-containing foods and 25 subjects of negative control group. This study aimed to determine whether there is a significant difference in the levels of N7-CAG between the two groups. DBS samples were extracted using the QIAamp DNA Mini Blood Kit and analyzed using Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS). Separation was performed using an Acquity UPLC BEH C18 column (2.1 mm × 100 mm; 1.7 µm), eluted a flow rate of 0.1 ml/min under an isocratic of mobile phase of 0.1% formic acid and acetonitrile. The bioanalytical method of N7-CAG in DBS with allopurinol as the internal standard by using UHPLC-MS/MS has been validated. The calibration curve range of N7-CAG obtained was 10-300 ng/ml with a coefficient of correlation of 0.997. The results of the analysis on 25 test group subjects showed that the concentration of N7-CAG ranged from 1.87 to 23.71 ng/ml, while the 25 subjects in the negative group ranged from 1.18 to 8.47 ng/ml. The results of the Mann Whitney test showed that there was a significant difference in the levels of N7-CAG between the test group and the negative control group with p value less than 0.001.

Evaluation and Characterization of Hard-Shell Capsules Formulated by Using Goatskin Gelatin.

Gelatin is used as an additive in medicine, food, and cosmetics. Gelatin from goatskin is a new excipient that has not been explored by researchers, including for hard-shell capsules. The aim of this study was to evaluate and characterize the hard-shell capsules produced from goatskin gelatin. The goatskin gelatin was extracted by an acid hydrolysis method, and the functional properties were investigated. Hard-shell capsules were then produced from goatskin gelatin, evaluated, and characterized. The gelatin extracted from goatskin had 56.9% ± 0.95 clarity and a pH of 5.11 ± 0.09, 97.51% ± 1.1 protein content, 9.23% ± 0.08 water content, 0.18% ± 0.07 ash content, 2.08% ± 0.35 fat content, gel strength of 298 ± 2.64 gbloom, and viscosity of 27.33 ± 2.07 mPs. The gelatin has met the requirements to be made into hard-shell capsules. The average weight of the hard-shell capsules produced was 96.9 mg with 8.69 standard deviation. The average size of the body and cap length was 18.84 ± 0.64 mm and 10.98 ± 0.30 mm, respectively. The results of capsule evaluation and characterization were as follows: the pH was 4.82 ± 1,27, water content was 10.03 ± 0.21, disintegration time was 4.02 ± 2.09 min, and there was no microbial growth. Thus, the capsules made have met the requirements and can be produced in a large quantity.

Development and validation of a UPLC-MS/MS method with volumetric absorptive microsampling to quantitate cyclophosphamide and 4-hydroxycyclophosphamide.

Cyclophosphamide (CP) is an anti-cancer alkylating prodrug, metabolized by CYP450 into its active metabolite 4-hydroxycyclophosphamide (4-OHCP). Its therapeutic effectiveness is determined by the 4-OHCP concentration. Several analytical methods in plasma and dried blood spots have been developed to analyze cyclophosphamide and 4-OHCP; however, there are many disadvantages. The objective of this study was to develop and validate the ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method by volumetric absorptive microsampling (VAMS) and 4-hydroxycyclophosphamide-d4 (4-OHCP-d4) as an internal standard. VAMS requires small sample volumes, and it is not affected by the hematocrit values; therefore, it is an efficient sampling method. The samples were derivatized with 5 µL semicarbazide hydrochloride (SCZ) and 25 µL of the resulting 4-OHCP-SCZ; 4-OHCP-d4-SCZ derivatives were absorbed by VAMS and extracted by protein precipitation. The optimum conditions were obtained using the Waters Acquity® UPLC BEH C18 (2.1 × 100 mm; 1.7 µm) column; flow rate 0.15 ml/min; mobile phase 0.01% formic acid and methanol; gradient elution mode for 6 min by positive electrospray ionization; and multiple reaction monitoring of m/z 260.7 > 140.0 for CP, 333.7 > 221.0 for 4-OHCP-SCZ, and 337.7 > 225.1 for 4-OHCP-d4-SCZ. The method met the validation requirements set by the FDA. The cyclophosphamide LLOQ value was 5 ng/mL, and the calibration curve range was 5-60,000 ng/ml. Furthermore, the 4-OHCP LLOQ value was 2.5 ng/ml, and the calibration curve range was 2.5-1,000 ng/ml.

Study of valproic acid liposomes for delivery into the brain through an intranasal route.

Intranasal drug transport through the olfactory route to the brain is an effective drug route for increased absorption and bioavailability of the drug. The objective of this study was to increase the penetration of valproic acid as an anticonvulsant into a delivery system comprising liposomes. Valproic acid liposomes were prepared by a thin-layer hydration technique using soybean phosphatidylcholine and cholesterol as the main ingredients. The formulations were evaluated for diameter size, entrapment efficiency (EE), zeta potential, polydispersity index, and morphology. ex vivo permeation using sheep nasal mucosa and in vivo efficacy were assessed by performing a pharmacokinetic study in Wistar albino rats following intranasal administration of the formulations in comparison with pure drug. The mean size particle of optimized liposomes ranged from 90 to 210 nm with a low polydispersity index (<0.5). The EE of optimized liposomes was between 60% and 85%, increasing the concentration of phosphatidylcholine added to the formula. Transmission electron microscopy observations (40,000×) showed that valproic acid liposomes have a spherical molecular shape and a particle size of below 250 nm. The ex vivo and in vivo results showed that liposomal formulations provided enhanced brain exposure. Among the formulations studied, Formula 4 (F4) showed greater uptake of valproic acid into the brain than plasma. The high brain targeting efficiency index for F4 indicated the preferential transport of the drug to the brain. The study demonstrated the successful formulation of surface-modified valproic acid liposomes for nasal delivery with brain targeting potential.

Single dose oral pharmacokinetic profile rubraxanthone in mice.

Rubraxanthone is a main active constituent of kandis (Garcinia cowa Roxb), has showed many biological activity including antimicrobial, anti hypercholesterolemic, antiplatelet, antioxidant, cytotoxic, and anti-inflammatory properties. To the best of our knowledge, no reports on the pharmacokinetics (PK) of this rubraxanthone have been published. The PK of rubraxanthone in mice was examined after a single oral dose of 700 mg/kg rubraxanthone suspension in virgin coconut oil. Plasma samples were collected at different time points and further analyzed using validated chromatographic method. Pharmacokinetic parameters were calculated from observed plasma concentration-time profile. The maximum concentration of rubraxanthone in plasma was discovered in 1.5 h. The peak plasma concentration (Cmax) was 4.267 µg/mL, and the area under the curve (AUCt0-t ∞ ) was 560.99 µg h/L, with a 6.72-hour terminal half-life (T1/2). The volume of distribution (Vd/F) was 1200.19 mL/kg and 1123.88 mL/h/kg clearance (Cl/F).

Analysis of Acrylamide and Glycidamide in Dried Blood Spot of Smokers Using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

INTRODUCTION: Acrylamide is a genotoxic substance that can be found in cigarette smoke. Acrylamide is metabolized by the CYP2E1 enzyme in the body to form glycidamides, an epoxide that is reactive to DNA and can form carcinogenic adducts. Therefore, exposure to acrylamide can potentially cause cancer. This study aims to analyze the levels of acrylamide and glycidamide in dried blood spot samples of smokers using propanamide as an internal standard and non-smokers as the control subjects. METHODS: Dried blood spot samples were extracted using the protein precipitation method and then analyzed by liquid chromatography-tandem mass spectrometry. Mass detection was performed using positive type electro spray ionization and multiple reaction monitoring type with m/z 72.0>55.02 for acrylamide, 88.1>45.0 for glycidamide, and 74.0>57.1 for propanamide as the internal standard. RESULTS: Acrylamide and glycidamide levels in the dried blood spot sample of smokers ranged between 3.91 -10.25 µg/mL and 1.006-3.58 µg/mL, respectively. Data of the non-smokers on acrylamide and glycidamide levels were 0.75-3.16 µg/mL and 0-0.91 µg/mL. DISCUSSION: The significant value of acrylamide and glycidamide between smokers and non-smokers was p < 0.05, which showed that there is a significant difference between acrylamide and glycidamide concentration in smokers and non-smoker subjects. The results of this study suggest that dried blood spots can be used to determine acrylamide and glycidamide levels in humans. Theoretically, acrylamide and glycidamide concentration should correlate to each other; however in reality, there are other factors (such as CYP2E1 polymorphism, dietary intake, etc) that can cause variation in their respective concentration.

Rectal Administration of Ibuprofen: Comparison of Enema and Suppository Form.

Ibuprofen is a widely used and well-tolerated analgesic and antipyretic. It is desirable to have a formulation with a rapid rate of absorption because it is required for rapid pain relief and temperature reduction. Previous studies have described the pharmacokinetic profiles of ibuprofen suppository and the mean peak times of ibuprofen suppository were around 1.8 hours, indicating a slower rate of absorption. The aim of this study is to compare the pharmacokinetic parameters of rectal administration of ibuprofen between enema and suppository form in order to provide evidence for the faster absorption rates of ibuprofen enema. This study was a phase-1 clinical study, open-label, randomized and two-way crossover with one-week washout period comparing the absorption profile of equal dose of ibuprofen administered rectally in two treatment phases: ibuprofen suppository and enema. Blood samples were collected post dose for pharmacokinetic analyses. Tmax was analyzed using a Wilcoxon matched paired test. A standard ANOVA model, appropriate for bioequivalence studies was used and ratios of 90% confidence intervals were calculated. This study showed that Tmax for ibuprofen enema was less than half that of ibuprofen suppository (median 40 min vs. 90 min, respectively; p-value=0.0003). Cmax and AUC0-12 for ibuprofen enema were bioequivalent to ibuprofen suppository, as the ratio of test/reference=104.52%, 90% CI 93.41-116.95% and the ratio of test/reference=98.12%, 90%CI 93.34-103.16%, respectively, which fell within 80-125% bioequivalence limit. The overall extent of absorption was similar to the both, which were all well tolerated. In terms of Tmax, Ibuprofen enema was absorbed twice as quickly as from ibuprofen suppository. Therefore it is expected that an ibuprofen enema may provide faster onset of analgesic and antipyretic benefit.

The correlation between the level of 3-hydroxypropyl mercapturic acid, CYP2B6 polymorphisms, and hematuria occurrences after cyclophosphamide administration and its bioanalytical methods: A systematic review.

BACKGROUND: Cyclophosphamide (CPA) is a cytotoxic prodrug that needs to be metabolized by cytochrome P450 enzymes, like CYP2B6. Unfortunately, CYP2B6 is a very polymorphic enzyme and can cause a change in 3-hydroxypropyl mercapturic acid (3-HPMA), the most found CYP metabolite in urine levels. Change in 3-HPMA levels can also indicate the level change in its precursor, acrolein, which is responsible for the hematuria incidence after CPA administration.This review's purpose is to obtain a conclusion about the optimal 3-HPMA analysis method in urine after the administration of cyclophosphamide using liquid chromatography-tandem mass spectrometry (LC-MS/MS) through literature review from previous studies. Also, this review was written to examine the relationship between levels of 3-HPMA in urine, polymorphisms of CYP2B6 enzymes, and the incidence of hematuria after cyclophosphamide administration in cancer patients. METHODS: Major databases, such as Universitas Indonesia's library database ScienceDirect, PubMed/Medline, Frontiers Media, and Google Scholar database, were used to find both published and unpublished studies without a time limit until 2020. Studies on pharmacokinetics, pharmacodynamics, drug therapy monitoring of cyclophosphamide, bioanalysis, and polymerase chain reaction (PCR) published in Indonesian and English were included. Meanwhile, non-related studies or studies written in other languages besides Indonesian and English were excluded. Two independent reviewers screened the titles, abstracts, and full-text manuscripts. Data obtained from eligible sources were used to answer the purpose of this review in a narrative form. RESULTS: The authors found 436 related studies from various databases and websites. Then, the authors narrowed it down into 62 pieces of literature by removing the duplicates and reviewing the abstracts and full-text manuscripts. Out of 62 sources, the authors found 30 studies that explained 3-HPMA analysis using LC/MS-MS, CYP2B6 polymorphisms, and hematuria occurrences. The authors used those 30 studies to build a conclusion regarding the purpose of this study. We strengthened the results with some additional information from the other 32 eligible sources. CONCLUSIONS: The authors conclude that according to literature searches from previous studies, the optimal 3-HPMA analysis method in urine after cyclophosphamide administration using LC-MS/MS is using triple quadrupole LC-MS/MS; source of positive ion electrospray ionization (ESI); mobile phase combination of 0.1% formic acid in water (A) - 0.1% formic acid in acetonitrile (90:10 v/v) (B); the Acquity® BEH C18 column (2.1 × 100 mm; 1.7 µm); injection volume of 10 µl; flow rate of 0.2 ml/minute; gradient elution method. Detection was carried out using mass spectrometry with m/z ratio of 222.10 > 90 for 3-HPMA and m/z 164.10 > 122 for n-acetylcysteine (NAC). The optimum sample preparation method is acidification and dilution ratio of 1:5 v/v. Also, there is a relationship between 3-HPMA levels, CYP2B6 polymorphisms, and the occurrences of hematuria after the administration of cyclophosphamide, which is a type of CYP2B6 polymorph, namely CYP2B6∗6, can increase cyclophosphamide hydroxylation so that it can increase the levels of acrolein and 3-HPMA, as its metabolites, and risk of hematuria. ETHICS AND DISSEMINATION: This research does not use human participants, human data, or human tissue for being directly studied for the review. Therefore, ethics approval and consent to participate are not applicable. REGISTRATION: This research has not been registered yet.

Development and Validation of the Quantification Method for Hydroxychloroquine in Volumetric Absorptive Microsampling (VAMS) Using High-Performance Liquid Chromatography-Photodiode Array.

Hydroxychloroquine is an antimalarial drug used for systemic lupus erythematosus, rheumatoid arthritis, and malaria treatment. However, hydroxychloroquine has several side effects such as ocular toxicity, neurotoxicity, gastrointestinal disorder, and also severe toxicity such as cardiotoxicity. Therefore, therapeutic drug monitoring of high dose or long-term use of hydroxychloroquine is needed. This study aims to obtain an optimum and validated analysis and preparation method for hydroxychloroquine in volumetric absorptive microsampling (VAMS) using the high-performance liquid chromatography-photodiode array detector based on the Food and Drug Administration guidelines (2018). Hydroxychloroquine quantification was performed using HPLC-PDA with Waters Sunfire™ C18 (5 µm; 250 × 4,6 mm) column. Mobile phase consists of acetonitrile-diethylamine 1% (65 : 35, v/v) (isocratic elution) and delivered at a flow rate of 0.8 mL/min throughout the 12 minutes run. Sample in VAMS is extracted by liquid-liquid extraction with ammonia 1% and n-hexane-ethyl acetate (50 : 50 v/v) as a extraction solvent. This method has successfully qualified the Food and Drug Administration (2018) parameters, with 2 ng/mL of LLOQ, range of calibration curve 2-6500 ng/mL, and coefficient of correlation 0.9993-0.9997.

Determination of O6-Methylguanine in dried blood spot of breast cancer patients after cyclophosphamide administration.

Cyclophosphamide is a nitrogen mustard class of drugs that are often used in cancer chemotherapy. However, the use of Cyclophosphamide in high doses over a long period has been shown to increase the risk of developing secondary cancer. This can be indicated by the formation of mutagenic DNA adducts, such as O6-Methylguanine. Therefore, this adduct can be used as a biomarker for secondary cancer in patients receiving Cyclophosphamide. Bio sampling was carried out by using the Dried Blood Spot (DBS) method, followed by DNA extraction by using QIAamp DNA mini kit, and acid hydrolysis to obtain O6-Methylguanine. Analysis of O6-Methylguanine was performed by using the UPLC-MS/MS instrument with the conditions developed by Vianney, Harahap, & Suryadi (2021). Partial validation was carried out before the analysis. The results obtained from the calibration curve, accuracy, and precision validation test met the FDA requirements. The analysis method was then implemented in 16 breast cancer patients who received the Cyclophosphamide regimen. The O6-Methylguanine was successfully detected and quantified in all of the samples in the range of 0.55-6.66 ng/mL. It shows that the O6-Methylguanine accumulation in cancer patients receiving Cyclophosphamide is very likely to occur and the analysis method proposed by Vianney, Harahap, & Suryadi (2021) is potential to be used for Therapeutic Drug Monitoring in this group of patients.

Volumetric Absorptive Microsampling as a New Biosampling Tool for Monitoring of Tamoxifen, Endoxifen, 4-OH Tamoxifen and N-Desmethyltamoxifen in Breast Cancer Patients.

INTRODUCTION: In this research, we used a volumetric absorptive microsampling (VAMS) technique to collect blood samples from the patients. A rapid and simple sample preparation method and LC-MS.MS assay was then developed and validated for the simultaneous analysis of tamoxifen and its three active metabolites. METHODS: VAMS extraction was performed in methanol by sonication-assisted extraction method for 25 min after 2 hof VAMS drying. Separation was carried out using Acquity UPLC BEH C18 column (2.1 x 100 mm; 1.7 µm), with a flow rate of 0.2 mL/min, and the mobile phase gradient of formic acid 0.1% and formic acid 0.1% in acetonitrile for 5 min. The multiple reaction monitoring (MRM) values were set at m/z 358.31>58.27 for N-desmethyltamoxifen, m/z 372.33>72.28 for tamoxifen, m/z 388.22>72.28 for 4-hydroxytamoxifen, m/z 374.25>58.25 for endoxifen, and m/z 260.26>116.12 for propranolol. RESULTS AND DISCUSSION: The lower limit of quantification value (LLOQ) was 2.50 ng/mL for tamoxifen, 2.50 ng/mL for endoxifen, 1.50 ng/mL for 4-hydroxitamoxifen, and 2.00 ng/mL for N-desmethyltamoxifen. Accuracy (%bias) and precision (%CV) were within 20% for LLOQ and 15% for other concentrations. There were no interference responses >20% of the LLOQ and 5% of the internal standard. The level of ion suppression in all analytes was less than 7%. The preparation system developed in this study successfully extracted more than 90% of analytes from the matrix with precision below 15%. Carryover was shown to be below 6% in all analytes. Stability of analytes in VAMS was demonstrated for up to 30 days, under room temperature storage in a sealed plastic bag with desiccant. This method was successfully applied to analyze tamoxifen and the metabolites level in 30 ER+ breast cancer patients.

Analysis of tamoxifen and its metabolites in dried blood spot and volumetric absorptive microsampling: comparison and clinical application.

This research was conducted to develop the Dried Blood Spot (DBS) and Volumetric Absorptive Microsampling (VAMS) method in the analysis of Tamoxifen (TAM) and its metabolites endoxifen (END), 4-hydroxytamoxifen (4-HT), and N-desmethyltamoxifen (NDT) using Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS). This method was then applied to monitor TAM and its metabolites in breast cancer patients. The UPLC-MS/MS method was developed and validated with propranolol as the internal standard. The recovery and matrix effects on DBS and VAMS were investigated. The validation requirements were fulfilled by the methodology of analysis and sample preparation described in this study. Both VAMS and DBS extraction recoveries were satisfactory, with low variability. Extraction recovery in the VAMS sample was found to be slightly higher than in the DBS sample. Sample stability in DBS and VAMS was demonstrated for up to 2 months. Both of these methods were successfully applied for the analysis of TAM and metabolites in clinical patients. The mean concentrations obtained from the two methods were not significantly different.