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"Penggunaan siklofosfamid sebagai agen kemoterapi menyebabkan beberapa efek samping berupa mual, muntah, leukopenia, anemia, dan sistitis hemoragik. Efek samping yang disebabkan oleh siklofosfamid perlu mendapatkan perhatian dan penanganan khusus karena dapat membahayakan pasien. Penelitian ini bertujuan untuk menganalisis prevalensi kejadian mual, muntah, leukopenia, anemia, dan sistitis hemoragik pada pasien kanker yang mendapatkan kemoterapi siklofosfamid di Rumah Sakit Kanker Dharmais Jakarta periode Juli 2012 - Juli 2013. Metode penelitian yang digunakan adalah cross-sectional dan pengambilan data retrospektif dilakukan dengan menggunakan data rekam medik pasien. Sampel adalah pasien yang mendapatkan kemoterapi siklofosfamid di Rumah Sakit Kanker Dharmais Jakarta periode Juli 2012 - Juli 2013. Pengambilan sampel sebanyak 79 sampel dilakukan dengan total sampling. Berdasarkan hasil penelitian didapatkan prevalensi efek samping pada pasien yang mendapatkan kemoterapi siklofosfamid di Rumah Sakit Kanker Dharmais Jakarta periode Juli 2012 - Juli 2013 adalah mual (69,6%), muntah (50,6%), leukopenia (96,2%), anemia (87,3%), dan sistitis hemoragik (13,9%). Muntah dipengaruhi oleh dosis (p=0,000).

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Some of adverse drug reactions such as nausea, vomiting, leucopenia, anemia, and hemorrhagic cystitis caused cyclophosphamide as chemotherapy agents. The side effects caused by cyclophosphamide should get special attention and treatment because it can harm the patient. This research aimed to analyze the prevalence of nausea, vomiting, leucopenia, anemia, and hemorrhagic cystitis of patients treated with cyclophosphamide chemotherapy in Dharmais Cancer Hospital Jakarta period July 2012 - July 2013. The research design was cross-sectional and retrospective by using the patient's medical record. Samples were cancer patients who received cyclophosphamide chemotherapy in Dharmais Cancer Hospital Jakarta period July 2012 - July 2013. Sampling was carried out as many as 79 samples with a total sampling. Based on the results, the prevalence of side effects of patients treated with cyclophosphamide chemotherapy in Dharmais. Cancer Hospital Jakarta period July 2012 - July 2013 were nausea (69.6 %), vomiting (50.6 %), leucopenia (96.2 %), anemia (87.3 %), and hemorrhagic cystitis (13.9 %). Vomiting was influenced by dose (p=0,000)."

"Siklofosfamid merupakan obat antikanker berupa pro-drug yang diaktifkan oleh enzim sitokrom P450 menjadi 4-hidroksisiklofosfamid 4-OHCP. Kadar 4-OHCP dalam tubuh dapat menggambarkan pembentukan fosforamid mustar yang dapat mengalkilasi DNA dan memberikan efek sitotoksik terhadap sel kanker. Analisis siklofosfamid dan 4-OHCP dilakukan pada Dried Blood Spot DBS 17 pasien kanker Rumah Sakit Kanker Dharmais yang diberikan rejimen siklofosfamid secara KCKUT-SM/SM sebagai upaya pemantauan terapi obat. Pengambilan darah dilakukan pada jam ke-2 dan ke-4 setelah pemberian kemoterapi melalui ujung jari. Hasil validasi metode bioanalisis parsial menghasilkan akurasi dan presisi intra hari dengan diff dan koefisien variasi KV tidak lebih dari 15 dan tidak lebih dari 20 pada konsentrasi LLOQ. Kurva kalibrasi yang linear didapat pada rentang 50-30.000 ng/mL untuk siklofosfamid dan 10-1000 ng/mL untuk 4- OHCP. Metode ini telah memenuhi syarat akurasi dan presisi intrahari sesuai European Medicines Agency EMEA tahun 2011. Hasil analisis pada 17 pasien kanker menunjukkan kadar siklofosfamid berkisar antara 6045,980 ng/mL hingga 37024,403 ng/mL dan 4-OHCP berkisar antara 33,155 ng/mL hingga 246,362 ng/mL. Hasil yang diperoleh dapat menjadi salah satu parameter pemantauan terapi siklofosfamid.

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Cyclophosphamide is an anticancer in the form of prodrug activated by cytochrome P450 enzyme into 4 hydroxycyclophosphamide 4 OHCP. 4 OHCP levels in the body can describe formation of phosphoramide mustard that can alkylate DNA and give cytotoxic effects to cancer cells. Analysis of cyclophosphamide and 4 OHCP was performed on 17 Dried Blood Spots DBS of cancer patients who received cyclophosphamide as chemotherapy regiment from Dharmais Cancer Hospital by KCKUT SM SM for therapeutic drug monitoring. Samples were taken at 2nd and 4th hours after drug administration with finger prick method.

The results of partial validation method produced intra day accuracy and intra day precision with diff and coefficient of variation CV were not more than 15 and not more than 20 in LLOQ concentration. Linear calibration curves were obtained in the range of 50 ndash 30,000 ng mL for cyclophosphamide and 10 1000 ng mL for 4 OHCP. This method has been fulfilled for intra day accuracy and precision according to European Medicines Agency EMEA Guidelines, 2011. The results of the 4 OHCP concentration analysis in 17 cancer patients showed that cyclophosphamide levels ranging from 6045.980 ng mL to 37024.403 ng mL and 4 OHCP was in the range of 33.155 ng mL to 246.362 ng mL. The results could be one of the monitoring parameters of cyclophosphamide therapy."

"Siklofosfamid adalah kemoterapi yang bekerja sebagai agen pengalkilasi dan merupakan prodrug sehingga membutuhkan aktivasi oleh enzim sitokrom P450 untuk berubah menjadi metabolit aktifnya, yaitu 4-hidroksisiklofosfamid 4-OHCP. Tujuan penelitian ini adalah untuk memperoleh metode optimum dan tervalidasi untuk analisis siklofosfamid dan 4-OHCP dalam dried blood spot menggunakan Kromatografi Cair Kinerja Ultra Tinggi-Tandem Spektrometri Massa KCKUT-SM/SM. Penelitian siklofosfamid dan 4-hidroksisiklofosfamid secara simultan ini dikembangkan pertama kalinya dalam sampel dried blood spot. Metabolit 4-OHCP bersifat tidak stabil dalam cairan biologis, sehingga prosedur derivatisasi dilakukan sebelum analisis, yaitu menggunakan semikarbazid hidroklorida. Pemisahan dilakukan pada kolom UPLC Class BEH C18 menggunakan fase gerak asam format 0,01 dalam air-metanol 50:50 dengan mode elusi isokratik pada laju alir 0,3 mL/menit selama 4 menit. Deteksi massa dilakukan pada Waters Xevo TQD dengan Electrospray Ionization positif untuk siklofosfamid, 4-OHCP, dan heksametilfosforamid sebagai baku dalam dengan nilai m/z berturut-turut adalah 260,968 > 139,976; 338,011 > 224,979; dan 180,17 > 92,08. Ekstraksi dilakukan menggunakan metode pengendapan protein dengan pelarut metanol ? ?asetonitril 2:1. Metode ini linear pada rentang 50-30.000 ng/mL untuk siklofosfamid dan 10-1.000 ng/mL untuk 4-OHCP dengan r berturut-turut adalah 0,9972 dan 0,9984. Nilai LLOQ untuk siklofosfamid dan 4-OHCP berturut-turut adalah 50 ng/mL dan 10 ng/mL. Nilai diff dan KV untuk akurasi dan presisi intra hari dan antar hari tidak melebihi 15 dan tidak melebihi 20 pada konsentrasi LLOQ. Secara keseluruhan metode ini telah memenuhi persyaratan validasi yang mengacu pada European Medicines Agency Guidelines 2011.

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Cyclophosphamide is a chemotherapy that acts as an alkylating agent and a prodrug that requires activation by the cytochrome P450 enzyme to convert into its active metabolite, 4 hydroxycycrophosphamide 4 OHCP. The purpose of this research is to obtain the optimum and validated method for the analysis of cyclophosphamide and 4 OHCP in dried blood spots using Ultra Performance Liquid Chromatography Tandem Mass Spectrometry UPLC MS MS. This simultaneous cyclophosphamide and 4 OHCP study was developed for the first time in dried blood spot sample. The 4 OHCP metabolite is unstable in biological fluids, so the derivatization procedure was performed prior to analysis, using semicarbazide hydrochloride. The separation was performed on UPLC Class BEH C18 column using mobile phase of 0.01 formic acid in water methanol 50 50 with isocratic elution mode at 0.3 mL minute for 4 min. Mass detection was performed on Waters Xevo TQD with Positive Electrospray Ionization for cyclophosphamide, 4 OHCP, and hexamethylphosphoramide as internal standard with m z values respectively are 260.968 139.976 338.011 224.979 and 180.17 92.08. Extraction was performed using protein precipitation method with methanol acetonitrile 2 1. This method was linear in the range of 50 30,000 ng mL for cyclophosphamide and 10 1,000 ng mL for 4 OHCP with r respectively are 0.9972 and 0.9984. The LLOQ values for cyclophosphamide and 4 OHCP were 50 ng mL and 10 ng mL. The value of diff and CV for accuracy and precision intra days and between days did not exceed 15 and did not exceed 20 of LLOQ concentrations. Overall this method has met the validation requirements that refer to European Medicines Agency Guidelines 2011."

"Development and Validation of Cyclophosphamide and4-Hydroxycyclophosphamide Quantification Method inVolumetric Absorptive Microsampling (VAMS) by LiquidChromatography - Tandem Mass Spectrometry

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Cyclophosphamide is an anticancer alkylating prodrug, metabolized by CYP450 into its active metabolite, named 4-hydroxycyclophosphamide (4-OHCP). Its therapeutic effectiveness is determined by the 4-OHCP concentration. Several analytical methods using plasma and Dried Blood Spot have been developed to analyze cyclophosphamide and 4-OHCP. However, there are lots of disadvantages. Therefore, this study was conducted to develop a validated cyclophosphamide and 4-OHCP analysis method with 4-hydroxycyclophosphamide-d4 (4OHCP-d4) as the internal standard in Volumetric Absorptive Microsampling (VAMS) using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry. VAMS requires small volume of sample, is not affected by hematocrit, and more efficient sampling process. Sample preparation was started by derivatization with 5 μL semicarbazide hydrochloride to overcome the instability of 4-OHCP and 4-OHCP-d4, which was absorbed by VAMS. Afterwards, 25 μL samples were absorbed into VAMS and extracted using the protein deposition method with methanol. The analysis was performed using a triple quadrupole Mass Spectrometry with positive electrospray ionization mode. The optimum conditions were obtained using the Acquity® UPLC BEH C18 column (2.1 x 100 mm; 1.7 μm); flow rate 0.2 mL/min; mobile phase 0.01% formic acid and methanol; gradient elution mode for 6 minutes; multiple reaction monitoring detection with m/z values 260.65>140.03 for cyclophosphamide, 333.65>221.04 for 4-OHCP-SCZ, and 337.71>225.05 for 4-OHCP-d4-SCZ. The method has met the validation requirements set by the FDA (2018). Cyclophosphamide LLOQ value was 5 ng/mL and the calibration curve range was 5 - 60,000 ng/mL. Furthermore, 4-OHCP LLOQ value was 2.5 ng/mL and the calibration curve range was 2.5 - 1,000 ng/mL."

"Demam neutropenia merupakan efek samping yang sering terjadi setelah kemoterapi. Demam neutropenia dapat menyebabkan penundaan dosis kemoterapi sehingga dapat mengurangi efektivitas terapi. Kejadian demam neutropenia paskakemoterapi dapat dicegah dengan pemberian Granulocyte-colony Stimulating Factor (G-CSF). Regimen kemoterapi yang digunakan dapat memengaruhi kejadian demam neutropenia. Selain itu, usia, stadium kanker, riwayat kemoterapi dan kadar hemoglobin sebelum kemoterapi merupakan faktor risiko demam neutropenia paskakemoterapi.Penelitian ini bertujuan untuk mengetahui perbandingan kejadian demam neutropenia regimen TAC (dosetaksel, doksorubisin, siklofosfamid) dengan profilaksis primer G-CSF dan regimen FAC (fluorourasil, doksorubisin, siklofosfamid) pada pasien kanker payudara di RSUP Dr. Hasan Sadikin Bandung periode Januari 2017-Juni 2019.Desain penelitian adalah cross sectional uji dua populasi. Jumlah sampel sebanyak 61 regimen TAC dan 102 regimen FAC. Kejadian demam neutropenia dianalisis menggunakan chi-square.Hasil penelitian menunjukkan kejadian demam neutropenia paskakemoterapi lebih banyak terjadi pada regimen TAC dengan profilaksis primer G-CSF dibandingkan dengan regimen FAC. Kejadian demam neutropenia 12 kali lebih banyak terjadi pada regimen TAC dengan GCSF dibanding regimen FAC. Usia, stadium kemoterapi, riwayat kemoterapi dan kadar hemoglobin sebelum kemoterapi secara statistik tidak signifikan memengaruhi kejadian demam neutropenia paskakemoterapi.

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Febrile Neutropenia is a common side effect of chemotherapy. Febrile neutropenia can cause delayed chemo doses that can reduce the effectiveness of therapy. The incidence of febrile neutropenia can be prevented by administering Granulocyte-colony Stimulating Factor (G-CSF). The chemotherapy regimen can affect the incidence of febrile neutropenia. In addition, age, stage of cancer, history of chemotherapy and prechemotherapy hemoglobin level are risk factors for febrile neutropenia.This study aimed to compare the incidence of febrile neutropenia between TAC (docetaxel, doxorubicin, cyclophosphamide) regimen with G-CSF primary prophylaxis and FAC regimen (fluorouracil, doxorubicin, cyclophosphamide) in breast cancer patients at RSUP Dr. Hasan Sadikin Bandung period January 2017 - June 2019.The study design was cross sectional test of two populations. The sample consisted of 61 TAC regimen and 102 FAC regimen. The incidence of febrile neutropenia were analyzed using chi-square.The results showed that the incidence of post-chemotherapy febrile neutropenia is more common in TAC regimen with G-CSF primary prophylaxis than FAC regimen. The incidence of neutropenia is 12 times more common in TAC regimens with G-CSF than FAC regimen. Age, stage of chemotherapy, history of chemotherapy and pre-chemotherapy hemoglobin levels did not statistically significantly influence the incidence of febrile neutropenia."

"Kemoterapi merupakan salah satu modalitas terapi kanker payudara. Kombinasi dari 5-fluorourasil, doksorubisin, dan siklofosfamid (FAC) adalah protokol kemoterapi yang paling banyak digunakan. Penelitian ini bertujuan untuk mengidentifikasi masalah terkait obat pada pasien kanker payudara yang menjalani kemoterapi FAC di Rumah Sakit Kanker Dharmais dalam periode Juli-Desember 2019. Penelitian dilakukan menggunakan metode observasional deskriptif dengan desain penelitian potong lintang (cross-sectional) menggunakan data restropektif. Masalah terkait obat yang diidentifikasi pada penelitian ini, meliputi dosis dan interaksi obat. Hasil penelitian menunjukkan insiden kanker payudara tertinggi pada kelompok umur 44-54 tahun (43,70%) dan penggunaan obat penunjang tertinggi terdapat pada ondansetron (27,17%). Masalah terkait obat dengan dosis yang tidak sesuai sebesar 9,24%, di mana 7,56% dosis terlalu rendah dan 1,68% dosis terlalu tinggi, dan interaksi obat sebesar 9,24%, dimana 25% kategori mayor, 69,44% kategori moderat, dan 5,56% kategori minor. Penatatalaksana kemoterapi FAC pada pasien kanker payudara berpotensi menyebabkan masalah terkaitobat. Oleh karena itu, perlu pemantauan terapi obat pada pasien agar masalah terkait obat dapat diminimalisasi sehingga keberhasilan terapi dapat tercapai.

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Chemotherapy is one of the modalities of breast cancer therapy. The combination of 5-fluorouracil, doxorubicin and cyclophosphamide (FAC) is the most widely use chemotherapy protocol. This study aims to identify drug-related problems in breast cancer patients undergoing FAC chemotherapy at Dharmais Cancer Hospital in the period July-December 2019. The study was conducted using descriptive observational methods with cross-sectional research designs using restropective data. Drug-related problems identified in this study included dosage and drug interactions.

The results showed the highest breast cancer incident showed in the age group 44-54 years old (43.70%) and the highest use of supportive therapy was found in ondansetron (27.17%). Percentage of inappropriate doses was 9.24% with 7.56% doses too low and 1.68% doses too high, and drug interactions by 9.24%, where 25% the major category, 69.44% the moderate category, and 5.56% the minor category. Management of FAC chemotherapy in breast cancer patients has the potential to cause drug-related problems. Therefore, it is necessary to monitor drug therapy in patients so that drug-related problems can be minimized andtherapeutic success can be achieved."

"ABSTRAKSiklofosfamid (CPA) merupakan obat sitotoksik golongan agen pengalkilasi yang telah terbukti efektif penggunaannya dalam kemoterapi kanker payudara dan berbagai jenis limfoma. Sebagai prodrug, siklofosfamid harus dimetabolisme terlebih dahulu oleh enzim sitokrom P450, salah satunya adalah CYP2B6 yang bersifat sangat polimorfik. Selain itu, siklofosfamid juga dapat menyebabkan efek samping berupa sistitis hemoragik yang ditandai dengan hematuria dan dapat berkembang menjadi kanker kandung kemih. Efek samping tersebut disebabkan oleh salah satu metabolit dari siklofosfamid, yaitu akrolein. Akrolein akan dimetabolisme kembali menjadi beberapa metabolit, salah satunya adalah asam 3-hidroksipropil merkapturat (3-HPMA) yang berada di urin dengan kelimpahan yang paling banyak. Oleh karena itu, sifat toksik akrolein dapat dimonitor dari kadar 3-HPMA dalam urin. Namun, polimorfisme enzim CYP2B6 juga perlu dianalisis karena enzim tersebut merupakan salah satu enzim pertama yang mengubah siklofosfamid menjadi metabolitnya, yaitu 4-hidroksisiklofosfamid, yang nantinya akan diubah menjadi metabolit turunan lain. Skripsi ini memuat hubungan antara kadar 3-HPMA dalam urin, polimorfisme CYP2B6, dan kejadian hematuria setelah pemberian siklofosfamid beserta metode bioanalisisnya. Berdasarkan penelusuran literatur yang dilakukan, diperoleh bahwa KCKUT-SM/SM tipe ESI positif dengan metode preparasi sampel pengasaman dan dilusi menghasilkan hasil analisis kadar 3-HPMA dalam urin setelah pemberian siklofosfamid yang optimal. Selain itu, terdapat tipe polimorf CYP2B6 yang dapat meningkatkan hidroksilasi siklofosfamid sehingga kadar 3-HPMA juga dapat meningkat. Risiko kejadian hematuria turut bertambah tinggi seiring meningkatnya kadar 3-HPMA dalam urin. Skripsi ini dapat digunakan sebagai pertimbangan tenaga medis dalam pemberian siklofosfamid untuk meningkatkan efektivitas dan keamanan terapi.

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ABSTRACT

Cyclophosphamide (CPA) is an alkylating agent cytotoxic drug that has been proved effective in breast cancer and many types of lymphoma chemotherapy. As a prodrug, cyclophosphamide needs to be metabolized by cytochromes P450 enzymes, like CYP2B6 which is a very polymorphic one. Cyclophosphamide can also cause hemorrhagic cystitis that defined by hematuria that can lead to bladder cancer. That adverse effect is caused by one of the metabolites of cyclophosphamide that is acrolein. Acrolein will be metabolized into some other metabolites, one of them is 3-hydroxypropyl mercapturic acid (3-HPMA) that has the biggest abundance in the urine. Thus, acrolein toxicity can be monitored from 3-HPMA concentration in urine. However, CYP2B6 polymorphisms also must be analyzed because CYP2B6 is one of the first enzymes that breakdowns cyclophosphamide into its metabolite, which is 4-hydroxycyclophosphamide, that also will be metabolized into some other derivatives. This thesis informs the correlation between 3-HPMA urine concentration, CYP2B6 polymorphisms, and hematuria occurrences after cyclophosphamide administration and its bioanalysis methods. After the literature review, I found that positive ESI mode LC-MS/MS with acidification and dilution sample preparation method produces an optimal result for the 3-HPMA urine concentration after cyclophosphamide administration. Also, there is a type of CYP2B6 polymorph that increases CPA 4-hydroxylation which can lead to the rising of 3-HPMA concentration. The risk of hematuria also increases with the rising of 3-HPMA concentration in urine. This thesis can be used by medical personnel as a consideration in cyclophosphamide administration to increase the effectiveness and safety of the therapy.

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"Siklofosfamid merupakan senyawa antineoplastik golongan pengalkilasi yang banyak digunakan untuk mengobati penyakit keganasan. Alkilasi yang terjadi pada posisi N7 basa guanin di kedua untai rantai DNA mengakibatkan kegagalan replikasi sel yang berguna untuk terapi kanker. Kesalahan posisi alkilasi, salah satunya pada posisi O6 basa guanin, ternyata dapat memberikan efek mutagenik dan bahkan karsinogenik, yang dapat memicu kanker sekunder. Oleh karena itu, addition product (adduct) yang terbentuk akibat alkilasi tersebut, yaitu O6-metilguanin, dapat menjadi penanda biologis terhadap risiko terbentuknya kanker sekunder. Pada penelitian ini, dilakukan identifikasi senyawa O6-metilguanin dalam darah pasien kanker yang mendapatkan siklofosfamid dalam regimen kemoterapi selama minimal 4 siklus. Sampel yang digunakan adalah DNA yang diisolasi dari darah pasien kanker. Isolat DNA kemudian dihidrolisis dan dianalisis menggunakan metode Kromatografi Cair Kinerja Tinggi penukar kation kuat dengan fase gerak amonium format 30 mM pH 3,95 - metanol (94:6), suhu kolom 30°C, laju alir 1,2 ml/menit, dengan sistem deteksi fluoresensi menggunakan panjang gelombang eksitasi 300 nm dan emisi 370 nm. Penentuan O6-metilguanin dalam sampel dilakukan dengan membandingkan data waktu retensi sampel dengan standar, yaitu menit ke 7,600 dengan batas deteksi sebesar 20,74 ng/ml (setara dengan 128813,52 μV/s). Sampling dilakukan terhadap 27 pasien, tetapi hanya 17 sampel DNA pasien yang dapat teranalisis dan O6-metilguanin terdeteksi dalam 1 sampel DNA pasien.

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Cyclophosphamide is one of alkylating agent which widely use in chemotherapy. Alkylation occurred in N7-guanine position in both DNA strand causes the cancer cell failed to replicate, hence gives the cytotoxic effects which is beneficial for the cancer therapy. Contrary, if the alkylation occurres in O6-guanine position, the drug gives mutagenic and carcinogenic properties which vulnerably leads to secondary cancer. Therefore, detection of the adduct formed, O6-methylguanine, is able to become a biomarker for the risk of secondary cancer?s development. In this research, O6-methylguanine was identified from patient which had been receiving cyclophosphamid in their chemotherapy for minimum 4 cycles. Patient?s DNA which isolated from their blood, were being hydrolized and identified. Analytical method which use in this research was High Performance Liquid Chromatography with strong cation exchange column, mobile phase consisted of 30 mM ammonium formate pH 3,95-methanol (94:6), flow rate 1,2 ml/min, column temperature 30°C and detected at excitation wavelength 300 nm and emission wavelength 370 nm. Standart of O6-methylguanine in samples was conducted with comparing retention time data from sample and standar, which was eluted in 7.600 minute and with limit of detection as 20,74 ng/ml (equals to 128813,52 μV/s). Sampling was conducted in 27 patients but only 17 patient?s DNA samples were able to be analyzed and O6-methylguanine was detected in 1 sample."

"Siklofosfamid merupakan senyawa pengalkilasi golongan oksazafosforin, berkhasiat sebagai antineoplastik yang mengalkilasi DNA sehingga sel kanker gagal bereplikasi. Siklofosfamid dapat memberikan efek terapi apabila sudah termetabolisme menjadi 4-hidroksisiklofosfamid (4-OHCP). Analisis 4-OHCP dilakukan pada 26 orang pasien kanker Rumah Sakit Kanker ?Dharmais? yang mendapatkan regimen siklofosfamid. Darah yang digunakan sebagai sampel diambil pada 0,5 dan 1 jam setelah pemberian kemoterapi masing-masing sebanyak 3 ml. Analisis 4-OHCP dilakukan setelah plasma diderivatisasi dengan semikarbazid HCl menggunakan kromatografi cair kinerja ultra tinggi ? tandem spektrometer massa. Metode ini telah divalidasi pada penelitian sebelumnya, Hasil validasi metode secara parsial menghasilkan akurasi dan presisi intra hari dengan % diff dan koefisien variasi (KV) tidak lebih dari 15% dan tidak lebih dari 20% pada konsentrasi LLOQ. Kurva kalibrasi yang linear didapat pada rentang 50 ? 15.000 ng/ml untuk siklofosfamid dan 5 ? 1.000 ng/ml untuk 4-OHCP. Metode ini telah memenuhi syarat akurasi dan presisi intra hari sesuai EMEA Guidelines. Hasil analisis kadar 4-OHCP pada 26 pasien kanker menunjukkan kadar 4-OHCP yang terukur berada pada rentang 5,25 ng/ml hingga 832,44 ng/ml. Hal ini menunjukkan bahwa pengobatan dengan siklofosfamid pada 26 pasien kanker tersebut sudah tepat dan diharapkan dapat menyembuhkan kanker.

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Cyclophosphamide is an alkylating agent from oxazaphosphorine compound, used as antineoplastic which alkylate the DNA cause the cancer cell fail to replicate. Cyclophosphamide is able to give therapeutic effect if it has been metabolized into 4-hydroxycyclophosphamide (4-OHCP). 4-OHCP analysis performed on 26 cancer patients in ?Dharmais? Cancer Hospital who get cyclophosphamide. Blood used as samples taken at 0,5 and 1 hour after administration of chemotherapy respectively 3 ml. Analysis of 4-OHCP done after plasma was derivatized with semicarbazide HCl by Ultra High Performance Liquid Chromatography ? Tandem Mass Spectrometry. This method has been validated in the previous research. Results of partial method validation produced intra-day accuracy and precision which % diff and coefficient of variation (CV) not more than 15% and not more than 20% at LLOQ concentration. A linear calibration curve produced in the range of 50 ? 15.000 ng/ml for cyclophosphamide and 5 ? 1.000 ng/ml for 4-OHCP. This method has been qualified for intra-day accuracy in accordance to EMEA Guidelines. Results of the analysis of 4-OHCP in 26 cancer patients showed the measured value of 4-OHCP are in the range of 5,25 ng/ml to 832,44 ng/ml. This suggests that treatment with cyclophosphamide in 26 cancer patients were appropriate and is expected to cure the cancer."

"Siklofosfamid (CP) merupakan salah satu obat kanker golongan agen pengalkilasi yang efektif digunakan untuk mengobati kanker payudara, limfoma non-Hodgkin, dan lain-lain. CP harus diubah menjadi metabolit aktifnya (4-hidroksisiklofosfamid/4-OHCP) untuk menghasilkan efek terapeutik. Siklofosfamid diubah menjadi 4-OHCP oleh beberapa enzim di hati, salah satunya sitokrom P450 2B6 (CYP2B6). CYP2B6 merupakan salah satu gen CYP yang paling bersifat polimorfik yang dapat memengaruhi regulasi transkripsional, ekspresi protein, dan kadar 4-OHCP dalam tubuh. Kadar 4-OHCP dapat menjadi parameter bahwa terapi yang diberikan efektif. Oleh karena itu, tujuan dari penelitian ini adalah untuk menentukan kecepatan hidroksilasi 4-OHCP dengan cara membandingkan konsentrasi 4-OHCP terhadap CP. Penelitian ini menggunakan 43 sampel Dried Blood Spot (DBS) pasien kanker payudara Indonesia yang terdapat CP dalam regimen terapinya. Darah pasien rata-rata diambil pada 2,23±0,38 jam (tmax CP) setelah pemberian kemoterapi. Sampel diekstraksi dengan pengendapan protein dan dianalisis menggunakan Kromatografi Cair Kinerja Ultra Tinggi Tandem Spektrometri Massa (KCKUT-SM/SM); kolom Acquity UPLC BEH C18 (2,1 x 100 mm; 1,7μm); suhu kolom 50°C; fase gerak asam format 0,01% - metanol dengan elusi gradien; laju alir 0,15mL/menit; volume injeksi 10 μL. Deteksi massa menggunakan ESI (+) dengan nilai m/z 260,65>140,03 untuk siklofosfamid, 33,65>221,04 untuk 4-OHCP-SCZ, dan 337,71>225,05 untuk 4-OHCP-d4-SCZ. Validasi parisal yang dilakukan memenuhi persyaratan FDA 2018. Metode ini linear pada rentang 5 – 60.000 ng/mL untuk CP dan 5 – 1000 ng/mL untuk 4-OHCP. Hasil Penelitian menunjukkan bahwa dari 43 pasien didapatkan rentang CP 2106,16 – 34386,90 ng/mL. dan 4-OHCP 24,85 – 995,071 ng/mL. Berdasarkan rasio 4-OHCP/CP, terdapat 53% (23 subjek) tergolong rapid metabolizer, dan 47% (20 subjek) tergolong poor metabolizer.

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Cyclophosphamide (CP) is an alkylating agent for anticancer and effective in treating breast cancer, non-Hodgkin lymphoma, and others. CP must be converted to its active metabolite (4-hydroxycyclophosphamide/4-OHCP) to produce a therapeutic effect. CP is converted to 4-OHCP by several enzymes in the liver, cytochrome P450 2B6 (CYP2B) is one of them. CYP2B6 is one of the most polymorphic CYP genes that can affect transcriptional regulation, protein expression, and the level of 4-OHCP in the body. The level 4-OHCP can be a parameter of whether the therapy is effective. Therefore, the purpose of this study is to determine the hydroxylation rate of 4-OHCP by comparing the level of 4-OHCP to CP. This study used a sample of 43 breast cancer patients Dried Blood Spot who contained CP in their regiment therapy which was taken in average time 2.23± 0.38 hours (tmax CP) after CP’s administration. Samples were extracted by protein precipitation method and analysed using Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS); Acquity UPLC BEH C18 column (2,1 x 100 mm; 1,7μm); temperature was 50°C; 0,01% formic acid - methanol as mobile phase with gradient elution for 6 minutes; flow rate was 0,15mL/minute; and injected volume 10 μL. Mass detection using a triple quadrupole with ESI (+) and multiple reaction monitoring detection with m/z values 260,65>140,03 for cyclophosphamide, 33,65>221.04 for 4-OHCP-SCZ, dan 337.71>225.05 for 4-OHCP-d4-SCZ. The partial validation performed has successfully met the validation requirements that refer to FDA 2018. This method was linear in the range of 5 – 60.000 ng/mL for CP and 5 – 1000 ng/mL for 4-OHCP. The result showed that from 43 patients, the CP levels ranged from 2106,16 – 34386,90 ng/mL. and 24,85 – 995,071 ng/mL for 4-OHCP. Based on the 4-OHCP/CP ratio, 53% (23 subjects) were classified as rapid metabolizers, and 47% (20 subjects) were classified as poor metabolizers."