Introduction

Cyclophosphamide is a cytotoxic drug that’s used for the therapy of many strong and hematologic neoplasms.¹ It may be used for single or mixture remedy for numerous indications corresponding to malignant lymphoma, acute and power lymphatic leukemia, strong tumors corresponding to breast most cancers, or as immunosuppressants. Cyclophosphamide remedy may cause extreme unintended effects corresponding to anemia, leukocytopenia, and thrombocytopenia, gonadal toxicity, and might even trigger secondary tumor improvement.² It is usually categorised as a gaggle 1 carcinogen compound (carcinogen in people) by the IARC.³

Cyclophosphamide belongs to the group of nitrogen mustard which is a bifunctional alkylating agent that damages DNA and type DNA adduct.⁴ Alkylation of DNA bases offers rise to the mutagenicity and carcinogenicity of the cyclophosphamide, particularly within the O6-guanine and N7-guanine positions. Alkylation in these positions causes crosslinks which point out cytotoxic properties.⁵ The formation of DNA adducts is the preliminary step within the strategy of carcinogenic results. Due to this fact, a technique is required to research DNA adduct from cyclophosphamide particularly O⁶-methylguanine as a secondary most cancers threat biomarker.

This research makes use of Dried Blood Spot samples for evaluation. Dried Blood Spot is likely one of the biosampling strategies that was accomplished utilizing finger prick and the blood was collected to fill a premarked circle on a filter paper.⁶ In comparison with earlier research that used venipuncture for blood sampling,⁷ DBS technique is much less invasive and solely requires a small quantity of blood thereby rising affected person consolation.⁸ As well as, the soundness of the pattern is best when utilizing the DBS technique in comparison with blood from veins.⁹

On this research, the event and validation of the analytical technique had been carried out utilizing UPLC-MS/MS with allopurinol as the inner normal. This research goals to acquire the optimum and validated analytical technique for measuring O⁶-methylguanine in Dried Blood Spot samples utilizing Extremely-Excessive-Efficiency Liquid Chromatography-Tandem Mass Spectrometry. Validation of this analytical technique refers back to the Meals and Drug Administration, 2018.¹⁰ This analysis is predicted to be helpful for the implementation of drug remedy monitoring particularly cyclophosphamide drug remedy.

Supplies and Strategies

Chemical and Reagents

O⁶-methylguanine, N⁷-methylguanine, adenine, and guanine had been bought from Sigma Aldrich (St. Louis, MO, USA). Inner Commonplace Allopurinol was obtained from Jiangsu Yew Pharm (Yixing, China). Reagents corresponding to formic acid, acetic acid, methanol, ethanol, and acetonitrile had been bought from Merck Co. Ltd. (Darmstadt, Germany). Ultrapure water from Sartorius Water Filter System. Human blood was obtained from The Indonesian Pink Cross (Jakarta, Indonesia). Perkin Elmer 226 paper from Perkin Elmer (Waltham, USA). The reagents for the DNA Isolation are Proteinase Ok, Buffer AL, Buffer AW1, Buffer AW2, and Buffer AE (QIAamp DNA Mini Kits, QIAGEN).

Preparation of Inventory and Working Commonplace Answer

A inventory resolution of O⁶-methylguanine and allopurinol was ready at 1.0 mg/mL by diluting them in methanol. A sequence of working normal options at acceptable focus ranges had been obtained by way of diluting every normal resolution with water containing 0.5% (v/v) formic acid. Calibration samples had been ready by diluting a working resolution utilizing entire blood to acquire a calibration vary of 0.5–20 ng/mL. High quality management samples had been ready at 1.5 ng/mL (QCL), 10 ng/mL (QCM), and 15 ng/mL (QCH) for O⁶-methylguanine by diluting working resolution in entire blood.

UPLC-MS/MS Situations

The analysis was carried out on an ACQUITY^TM UPLC system (Waters Corp., Milford, MA, USA) and a Xevo TQD Triple Quadrupole mass spectrometer (Waters Corp., Manchester, UK) geared up with optimistic electrospray ionization (ESI⁺). All information had been acquired in centroid mode by the MassLynx^TMNT4.1 software program and analyzed by the QuanLynx^TM program (Waters Corp., Milford, MA, USA). The analyte was separated on the Acquity^® UPLC BEH C₁₈ column (1.7 µm, 100 mm x 2.1 mm, Waters Corp., Milford, MA, USA). The cell part was 0.05% formic acid resolution and acetonitrile; movement charge 0.1 mL/min; autosampler temperature at 8°C; and the injection quantity was 10 µL. The gradient elution was used for six minutes and proven in Desk 1.

The mass spectrometric detector parameters had been optimized and set as follows: capillary voltage of three.50 kV, nitrogen desolvation temperature as 349°C with a movement charge of 643 L/h, column temperature of 40°C, and degasser stress of 0.69 psi. The cone voltage was 32 V for O⁶-methylguanine, 38 V for N⁷-methylguanine, 40 V for adenine, 35 V for guanine, and 35 V for allopurinol as IS. The detector was carried out in optimistic ion mode obtained by optimistic mode of electrospray ionization (ESI⁺) approach and quantification was acquired with a number of response monitoring (MRM) with ion transition at 165.95 → 149 and 165.95 → 134 for O⁶-methylguanine, m/z 165.95 → 149 and 165.95 → 124 for N⁷-methylguanine, m/z 135.9 → 118.95 for adenine, m/z 151.9 → 134.95 for guanine and m/z 136.9 → 110 for allopurinol as IS.

Preparation of Pattern in Dried Blood Spot

Blood samples had been obtained from The Indonesian Pink Cross (Jakarta, Indonesia) and had been reviewed and authorised by the Analysis Ethics Committees of “Dharmais” Most cancers Hospital, Jakarta 11420, Indonesia (No.023/KEPK/II/2020). Calibration and high quality management samples had been ready by pipetting 50 μL of entire blood containing O⁶-methylguanine onto the Perkin Elmer 226 paper and dried at room temperature for two h. DBS discs had been minimize and inserted right into a microtube. The IS resolution, allopurinol, is made to a focus of 1 μg/mL and 20 μL was added. Then, the pattern was extracted utilizing the QIAamp DNA Mini Equipment. DNA extraction procedures check with the QIAamp DNA Mini and Blood Mini Handbook¹¹ as follows:

• Dried Blood Spot pattern in a 1.5 mL microcentrifugation tube was added 180 µL of the ATL buffer and incubated at 85°C. Then, 20 µL of proteinase Ok resolution was added and incubated at 56°C. After that, 200 µL of the AL buffer was added to the pattern and incubated at 70°C.
• Samples had been added with 200 µL ethanol (96–100%) and the combination is rigorously transferred into the QIAamp mini spin column. QIAamp mini spin column was used on this extraction consisted of a designed silica layer that may entice the DNA on it when centrifuged.
• Then, AW1 and AW2 buffer was added to separate protein from DNA, subsequently it elevated the purity of DNA.
• Lastly, the DNA on the silica layer was eluted utilizing AE buffer and incubated at room temperature. The outcomes of DNA extraction might be saved at −20°C.

The DNA resolution was combined with the identical quantity of ultrapure water and 90% formic acid. Then, the answer is heated at 85°C for 60 minutes. After that, the answer is cooled to room temperature and able to be injected into UPLC-MS/MS.

Methodology Validation

Validation of this analytical technique was assessed together with Decrease Restrict of Quantification (LLOQ), calibration curve, selectivity, accuracy and precision, restoration, carryover, dilution integrity, matrix impact, and stability in keeping with the Meals and Drug Administration (FDA)Bioanalytical Methodology Validation Steering for Trade.¹⁰

Decrease Restrict of Quantification

LLOQ was decided utilizing clean samples and the bottom focus of analytes in samples that may nonetheless be analyzed quantitatively. The analyte response on LLOQ should be 5 occasions better than the analyte response on the clean pattern. The accuracy and precision of the analyte response ought to be lower than ± 20%.

Calibration Curve

The calibration curve was examined utilizing normal options which might be ready on the identical organic matrix because the research samples together with clean samples, zero calibrator, and not less than 6 different focus factors. The calibration curve acceptance standards are that the analyte focus isn’t greater than ± 15% in any respect concentrations aside from LLOQ and the analyte focus isn’t greater than ± 20% at LLOQ focus. A minimum of 75% of the calibration requirements with a minimal of 6 concentrations should meet these necessities.

Selectivity

The selectivity of the analytical technique was evaluated utilizing clean and LLOQ focus. The requirement for selectivity is the height response space of the analyte within the clean matrix obtained not more than ± 20% of the LLOQ peak space and not more than 5% within the inner normal.

Accuracy and Precision

Accuracy and precision had been evaluated by assessing repeat evaluation at 4 focus ranges within the measurement vary, particularly, LLOQ, QCL, QCM, and QCH over three consecutive days. The accuracy (%diff) and precision (%CV) ought to be inside ± 20% for LLOQ and ought to be inside ± 15% for QC samples.

Restoration

The restoration values had been obtained at three QC ranges (QCL, QCM, and QCH) by evaluating the outcomes of extracted samples with spiked post-extraction samples. Restoration values (%) would not have to be 100%, however the analytical, and normal restoration should be constant and reproducible.

Carryover

Carryover was evaluated by injecting clean samples after samples with excessive concentrations or calibration requirements on the higher restrict of quantification (ULOQ). Carryover shouldn’t be better than 20% of the analyte response on the decrease restrict of quantification (LLOQ) focus and never better than 5% for the inner normal.

Dilution Integrity

The dilution integrity was assessed by mixing the matrix with the analyte on the focus above the ULOQ and diluting the pattern with a clean matrix. The dilution integrity is acknowledged to satisfy the necessities if the accuracy and precision are usually not greater than ± 15%.

Matrix Impact

Matrix impact was evaluated at QCL and QCH by evaluating the outcomes of spiked post-extraction samples and normal options containing analyte at equal concentrations. The coefficient of variation (%CV) of the matrix impact mustn’t better than ±15%. The standardized matrix issue values with the inner normal ought to receive the acceptance vary of 0.8 to 1.2.¹²

Stability

The steadiness examined contains inventory resolution stability, autosampler stability, short-term stability, and long-term stability. Inventory resolution stability of O⁶-methylguanine and allopurinol was evaluated within the quick time period at room temperature and long run at −4°C. The %diff worth of the inventory resolution stability check mustn’t exceed ±10%.¹³ The opposite stability check was evaluated utilizing QCL and QCH focus. Autosampler stability was examined at autosampler temperature, short-term stability at room temperature, and long-term stability was examined on the freezer −20°C. The accuracy (%diff) and %CV at every stage mustn’t exceed ±15%.

Outcomes and Dialogue

Extremely delicate and selective analytical technique is required for the willpower of DNA adduct as a result of it’s usually present in a really small stage. Due to this fact, this research used UPLC-MS/MS because the instrument chosen. The UPLC-MS/MS is taken into account as your best option for supporting bioanalytical research because of excessive sensitivity, selectivity, and rapidity.

Methodology Improvement

Optimization of Mass Situation

Mass spectrometric circumstances had been tuned in optimistic ionization mode associated to analyte fundamental properties. The chemical construction is proven in Determine 1. The spectra confirmed a high-intensity sign at m/z 165.95 → 149 and 165.95 → 134 for O⁶-methylguanine, m/z 165.95 → 149 and 165.95 → 124 for N⁷-methylguanine, m/z 135.9 → 118.95 for adenine, m/z 151.9 → 134.95 for guanine and m/z 136.9 → 110 for allopurinol as IS, respectively. The fragmentation spectrum of O⁶-methylguanine and N⁷-methylguanineare is proven in Determine 2. The capillary voltage used was 3.50 kV, nitrogen desolvation temperature was 349°C with a movement charge of 643 L/h, column temperature of 40°C, and the degasser stress of 0.69 psi. The cone voltage was 32 V, 38 V, 40 V, 35 V and 35 V for O⁶-methylguanine, N⁷-methylguanine, adenine, guanine, and allopurinol, respectively.

Determine 1 O6-methylguanine chemical construction.

Determine 2 Fragmentation spectrum of (A) O6-methylguanine and (B) N7-methylguanine.

Optimization of Cell Section Mixture

The collection of the cell part mixture was carried out in 4 variations, particularly, 0.05% acetic acid resolution – acetonitrile, 0.05% acetic acid – methanol, 0.05% formic acid – acetonitrile, and 0.05% formic acid – methanol. On this research, a mixture of 0.05% formic acid – acetonitrile was chosen as a result of it produced a greater chromatogram with the most important space.

Optimization of Cell Section Composition

The collection of the composition of the cell part is carried out utilizing a mixture of 0.05% acetic acid – acetonitrile which is split into 4 composition variations, particularly 95:5, 90:10, 80:20, and 50:50. Primarily based on the outcomes, the composition of the cell part 95:5 produces the most important space. Due to this fact, composition 95: 5 was chosen to be used within the evaluation.

Optimization of Stream Charge

On this experiment, variations within the movement charge are 0.1 mL/min, 0.2 mL/min and 0.3 mL/min. Primarily based on the outcomes, rising the movement charge has been proven to speed up the retention time of the analyte. Nevertheless, the sooner movement charge can enhance column stress which causes the column to turn into broken rapidly. The usage of a movement charge of 0.1 mL/min offers the best peak space even with an extended retention time of three.47 min. Due to this fact, variations within the movement charge of 0.1 mL/min had been chosen to be used on this research.

Optimization of Cell Section Gradient Elution

Gradient elution was often accomplished to extend the elution power which might produce a greater peak form and a bigger peak space response. On this experiment, a gradient elution check was carried out utilizing 2 variations as proven in Tables 1 and a couple of. The usage of a gradient elution produces an excellent chromatogram with a sharper peak form in comparison with isocratic circumstances. Due to this fact, the gradient elution profile 2 was chosen.

Desk 1 The First Gradient Elution Profile

System Suitability Take a look at

After acquiring the optimum evaluation circumstances, a system suitability check is then carried out to make sure that the programs are working correctly and are prepared for use for evaluation. On this research, the height space coefficient of variation (%CV) outcomes had been 0.68% for O⁶-methylguanine and 1.0% for allopurinol. The outcomes additionally confirmed a % CV worth of 0.14% for the retention time of the 2 compounds. This reveals that the system can be utilized to start out the evaluation and has fulfilled the necessities that %CV worth does no more than 6%.¹⁴

Optimization of Pattern Preparation

Optimization of DBS pattern preparation is completed by optimizing the drying time and recognizing quantity. On this research, optimization of the drying time of DBS samples was evaluated with time variations of 60 min, 120 min, and 180 min. From the experiments carried out, it’s identified that 120 min is enough time and supplies the best response space of chromatogram. Due to this fact, 120 min is set because the optimum drying time for DBS samples. After drying time is optimized, the recognizing quantity is optimized to get the optimum blood quantity that should be noticed on DBS paper. Optimization is evaluated utilizing three variations within the recognizing quantity of 30, 40, and 50 µL. Primarily based on the outcomes, the response of the height space of the 50 µL offers the best outcomes. Due to this fact, a noticed quantity of fifty µL was chosen because the optimum noticed quantity.

Methodology Validation

Decrease Restrict of Quantification

LLOQ determines the sensitivity of a technique and must be decided throughout technique improvement. LLOQ check for O⁶-methylguanine at a focus of 0.5 ng/mL resulted in a worth of %diff which ranged from −8.90% to 11.40% with a %CV of 8.95%. This information is proven in Desk 3. Primarily based on these outcomes, it’s identified that the accuracy and precision of the focus of 0.5 ng/mL meet the LLOQ necessities on FDA, 2018. This LLOQ focus was the identical as earlier research that carried out O⁶-methylguanine evaluation in blood samples.⁷ This reveals that Dried Blood Spot samples can present the identical sensitivity as blood samples.

Desk 3 The Accuracy and Precision from LLOQ of O6-Methylguanine

Calibration Curve

The calibration curve was produced from a focus of 0.5 ng/mL to twenty ng/mL with 6 focus ranges, clean, and nil samples. The calibration curve obtained was linear and meets the necessities of the correlation coefficient (r) worth better than 0.98.¹⁵ The outcomes of the calibration curve experiment confirmed that the %diff obtained fulfilled the necessities. Information of O⁶-methylguanine inter-day calibration curves are proven in Desk 4.

Desk 4 Information of Inter-Day Calibration Curve of O6-Methylguanine

Selectivity

Selectivity is a check parameter evaluated to make sure that the tactic used can measure analytes precisely. Primarily based on outcomes, the selectivity check was fulfilled the FDA necessities with %interference of the analyte between 8.64–13.54% and %interference of allopurinol between 1.329–2.022%. The chromatograms of clean, LLOQ, and QC samples are proven in Determine 3.

Determine 3 Consultant UPLC-MS/MS chromatograms of O6-methylguanine and allopurinol in (A) clean DBS; (B) DBS with analyte at LLOQ; (C) QCL; (D) QCM; (E) QCH.

Accuracy and Precision

Accuracy and precision exams had been carried out utilizing 4-level concentrations, particularly, LLOQ, QCL, QCM, and QCH. The intraday accuracy worth of O⁶-methylguanine ranged from 91.99–106.29% with %CV values ≤4.61%. The interday accuracy worth ranged from 96.23–109.45% with %CV values ≤4.97%. Primarily based on the outcomes, the accuracy and precision of O⁶-methylguanine meet the necessities with the worth of %diff and %CV at LLOQ concentrations don’t greater than 20% and the worth of %diff andpercentCV at different concentrations than LLOQ don’t greater than 15%. In contrast with earlier research,⁷ this research confirmed homogen information with decrease %CV worth. The intra- and inter-day accuracy and precision are proven in Desk 5.

Desk 5 The Intra- and Inter-Day Accuracy and Precision of O6-Methylguanine

Restoration

The restoration check is carried out to see the extraction effectivity on the pattern. In accordance with FDA 2018, the worth of restoration in evaluation utilizing a organic matrix doesn’t must be 100%. Nevertheless, it must be constant and reproducible Primarily based on the check, the typical restoration worth for O⁶-methylguanine is 82.62% at QCL focus, 82.50% at QCM focus, and 83.29% at QCH focus. As well as, the check outcomes additionally confirmed %CV for the concentrations of QCL, QCM, and QCH of three.97%, 2.71%, and 5.90%, respectively. The common restoration worth obtained for the usual in allopurinol is 81.10% with a %CV of two.09%.

Carryover

Carryover is a parameter that’s examined to find out the provision of analytes in clean samples after injecting excessive focus analytes (ULOQ). Primarily based on the outcomes, the worth of carryover was 11.12% to 12.65% for O⁶-methylguanine and 1.30% to 1.68% for the inner normal allopurinol. These outcomes point out that the carryover of O⁶-methylguanine and allopurinol meets the 2018 FDA necessities.

Dilution Integrity

Dilution integrity exams confirmed the worth of %diff is obtained between −3.70% and 14.62% with a %CV worth of two.82% for the 2QCH focus, 6.39% for the QCH focus, and three.90% for the ½QCH focus. The outcomes point out that it meets the dilution integrity necessities with the worth of %diff and %CV don’t exceed ±15%.

Matrix Impact

The common matrix issue obtained was 95.69% for QCL focus and 97.30% for QCH focus with CV values for QCL and QCH, particularly, 1.96% and 1.11%. The outcomes of testing the matrix impact on the inner normal allopurinol give a median matrix issue worth of 89.83% with %CV 4.60%. There may be little ion suppression from the matrix in opposition to the inner normal. That is as a result of competitors between the matrix and the inner normal within the strategy of including cost within the cell part. The outcomes of standardized normalized matrix components obtained had been 1.07% for the focus of QCL and 1.09% for the focus of QCH with the values of CV for QCL and QCH, respectively, 4.20% and 5.03%.

Stability

Inventory options of O⁶-methylguanine and allopurinol had been secure for twenty-four h in room temperature and 30 d within the fridge (−4°C). The steadiness check outcomes of O⁶-methylguanine and allopurinol confirmed in Desk 6. The information point out that O⁶-methylguanine and allopurinol are secure sufficient throughout pattern preparation and storage circumstances.

Desk 6 The Stability Take a look at Outcomes of O6-Methylguanine

Conclusion

In conclusion, the tactic for measuring O⁶-methylguanine in Dried Blood Spot was efficiently developed and validated. In contrast with the beforehand used technique,⁷ this technique has improved with a much less invasive biosampling technique and a smaller quantity of blood samples. The tactic supplies a speedy, delicate, and selective evaluation of O⁶-methylguanine utilizing UPLC-MS/MS with a linear focus vary between 0.5–20 ng/mL.

Ethics

This research was performed in accordance with the Declaration of Helsinki.

Acknowledgments

The authors acknowledge the monetary assist obtained from the Directorate of Analysis and Group Providers (DRPM) Universitas Indonesia, Depok, Indonesia with grant quantity NKB-1272/UN2.RST/HKP.05.00/2020, for the monetary assist of this analysis. This research was reviewed and authorised by the Analysis Ethics Committees of “Dharmais” Most cancers Hospital, Jakarta 11420, Indonesia (No.023/KEPK/II/2020).

Disclosure

The authors report no conflicts of curiosity. The authors alone are liable for the content material and writing of this text.

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