Kurumuş kan örneklerinde yasadışı maddelerin kromatografik tayini için yöntem geliştirilmesi
Küçük Resim Yok
Dosyalar
Tarih
2021
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi, Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/embargoedAccess
Özet
Adli toksikolojik amaçlı yapılan madde testlerinde kan, geniş analitik yöntem seçenekleri ve farmakolojik yorumunun daha kolay yapılabilmesi gibi nedenlerden dolayı en sık kullanılan biyolojik materyaldir. Kan örnekleri ile kişinin etkisi altında olduğu maddelerin tespiti yapılabilir, yakın geçmişe (saatler, günler) dair veriler edinilebilir. Kanda madde analiziyle ilgili yaşanan kısıtların önüne geçebilmek için kapiler kan örneklerini kurutarak (DBS) analiz teknikleri geliştirilmiştir. Örnekleme, kurutulan örneklerin taşınması, depolanması yönünden DBS oldukça pratik bir tekniktir. Bu teknik, örneklerin uzun süre bozulma ve hemoliz riski içermemesi gibi üstünlükleri de taşımaktadır. Bu tez çalışması, farklı sorbentler kullanılarak kurumuş kan örneği tekniği ile esrar, amfetamin, 3,4-metilenedioksi-N-metilamfetamin (MDMA), morfin ve kokain tayini için kromatografik yöntemler geliştirilmesi amaçlanmıştır. Bu kapsamda, kurumuş kan örneği çalışma prensibine uygun elektrolif çekim tekniği ile sentezlenmiş nanolifli sorbentler geliştirilerek kanda yasadışı madde tayinine uygunluğunun araştırılması hedeflenmiştir. Sıvı kromatografi ardışık kütle spektrometresi (LC-MS/MS) metodu kullanılarak THC-COOH, amfetamin, MDMA, morfin ve benzoilegonine ait optimum kromatografik koşulları belirlenmiştir. Kan örnekleri ve kurumuş kan örnekleri (Whatman 903) için iki ayrı matriks kullanılarak analitik yöntem validasyonu yapılmıştır. Elektrolif çekim yöntemi ile lif çekim çözeltisi yüksek gerilime maruz bırakılarak, polimerlerin elektriksel olarak yüklenmesi sağlanmış, selüloz ve termoplastik poliüretan (TPU) içeren 5 farklı nanolif sentezlenmiştir. Gerçek örnekler ve standart madde katımlı örneklerle yapılan analizlerde uygun materyal seçimi için yapılmıştır. 50 µL kan örneği üzerine 1 mL çözgen eklenip, sıvı-sıvı ekstraksiyon işlemi yapılmıştır. Oluşan organik faz temiz bir ependorfa aktarılmış ve kuruluğa kadar azot altında uçurulmuştur. Oluşan tortular 150 µL mobil faz karşımında çözülerek viallenmiş ve LC-MS/MS'e enjeksiyon yapılmıştır. DBS örnekleri ise, 50 µL kan damlatılıp 2 saat oda sıcaklığında kurutulmuş, oluşan kurumuş lekeler kesilerek 1 mL ekstraksiyon çözgeni içine aktarılmıştır. 30 dk ultrasonik banyo sonrasında kağıt/sorbent ortamdan alınarak kan örneği için yapılan prosedür uygulanmıştır. Birçok farklı çözgen ve çözgen karışımı denenmiş metanol-asetonitril 40:60 (v/v) oranında en uyumlu sonuçlar elde edilmiştir. Geliştirilen analitik yöntemlerde her bir analit bulunduğu örnek içinde ayırt edilebilmekte, kalibrasyon eğrisi korelasyon katsayısı her bir analit için >0.99'dur. Her bir analitin düşük (25 ng/mL), orta (100 ng/mL) ve yüksek (200 ng/mL) derişimlerde ±%20 gerikazanım aralığında ve korelasyon katsayısının %20'yi aşmadığı bu şekilde doğru, kesin ve tekrarlanabilir valide bir yöntem geliştirilmiştir. Kanda morfin 6.7, amfetamin 16.7, MDMA 22.7, benzoilekgonin 5.3, THC-COOH 25 ng/mL, DBS'de morfin 17.8, amfetamin 20.8, MDMA 22.7, benzoilekgonin 6.9, THC-COOH 25 ng/mL saptama alt sınırı değerlerine sahiptir. 1, 10 ve 30 gün için yapılan kararlılık çalışmasında kan ve DBS'de her bir analitin kararlı olduğu belirlenmiştir. Yapılan karşılaştırma çalışması sonucunda kan örnekleri ve DBS örneklerinin sonuçlarının birbiri ile uyumlu olduğu istatistiksel olarak belirlenmiştir (n=58, r=0.949). Standart olarak satışı bulunan Whatman 903 filtre kağıdının 48 saatte üretilmiş selüloz ve selüloz-TPU nanolifi ile en yakın sonuçlar verdiği belirlenmiştir. Kan örneklerini kurutmak için kullanılan sorbentler veya kartlar geliştirilmekte olup, ülkemizde henüz bu amaçla yapılan üretimi bulunmamaktadır. Bu geliştirilen nanolifler sayesinde; yurtdışından temin edilen bir malzemenin ülkemizde geliştirilmesi için ön çalışmalar tamamlanmıştır. Daha etkin, kullanım amacına yönelik, verimli bir ürün ya da analitlere özgü ürünler sentezlenip adli toksikolojik çalışmalarda yeni bir alan oluşturabilir.
In forensic toxicological substance analysis, blood is the most frequently used biological material due to wide analytical method options and easier pharmacological interpretation. With blood samples, the substances under the influence of the person can be determined, and data on the recent past (hours, days) can be obtained. In order to avoid the limitations experienced in blood substance analysis, analysis techniques by dried blood samples (DBS) have been developed. DBS is a very practical technique in terms of sampling, transportation, and storage of dried samples. In addition, it has advantages such as the long-term deterioration of the samples and the risk of haemolysis. This thesis, it is aimed to make a chromatographic determination of cannabis, amphetamine, 3,4-methylenedioxy-N-methylamphetamine (MDMA), morphine, and cocaine with DBS technique using different sorbents. In this context, it is aimed to investigate their suitability for the determination of illegal substances in the blood by developing nanofiber sorbents developed with the electrospinning technique in accordance with the working principle of DBS. Optimum chromatographic conditions of THC-COOH, amphetamine, MDMA, morphine and benzoylecgonine were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analytical method validation was performed using two different matrices for blood and dried blood samples (Whatman 903). With the electrospinning method, the fiber spinning solution was exposed to high voltage, polymers were electrically charged, and 5 different nanofibers containing cellulose and thermoplastic polyurethane (TPU) were synthesized. In the analyzes made with authentic blood samples and standard addition samples, studies were carried out for the selection of appropriate materials. 1 mL solvent was added to a 50 µL blood sample and liquid-liquid extraction was performed. The organic phase was transferred to a clean Eppendorf and evaporated under nitrogen to dryness. The residues were dissolved in 150 µL of mobile phase mixture and vials were injected to LC-MS/MS. DBS samples were dried at room temperature for 2 hours by dripping 50 µL of blood, and the dried spots were cut and transferred into 1 mL of extraction solvent. After 30 minutes of ultrasonic bath, the paper/sorbent was removed from the solvent and the procedure for blood sample was applied. Many different solvents and solvent mixtures were performed, and the most compatible results were obtained at the methanol-acetonitrile ratio of 40:60 (v/v). In the analytical methods developed, each analyte can be distinguished within the sample it contains, and the calibration curve correlation coefficient is >0.99 for each analyte. It is an accurate, precise, and reproducible validated method whereby each analyte is in the ±20% recovery range at low (25 ng/mL), medium (100 ng/mL), and high (200 ng/mL) concentrations and the correlation coefficient does not exceed 20%. The lower limit of quantification was for morphine 6.7, amphetamine 16.7, MDMA 22.7, benzoylecgonine 5.3, THC-COOH 25 ng/mL in blood, morphine 17.8, amphetamine 20.8, MDMA 22.7, benzoylecgonine 6.9, THC-COOH 25 ng/mL in DBS. In the stability study for 1, 10, and 30 days, each analyte was determined to be stable in blood and DBS. As a result of the comparison study, it was determined that the results of blood samples and DBS samples were compatible with each other (n=58, r=0.949), statistically. It has been determined that Whatman 903 filter paper gives the closest results with 48 hours-produced cellulose and cellulose-TPU nanofiber. Sorbents or cards used to dry blood samples are being developed and they are not yet produced for this purpose in our country. Preliminary studies have been completed for the development of a material supplied from abroad in our country for these developed nanofibers. A more effective, intended use, efficient product or products specific to analytes can be synthesized and create a new field in forensic toxicological studies.
In forensic toxicological substance analysis, blood is the most frequently used biological material due to wide analytical method options and easier pharmacological interpretation. With blood samples, the substances under the influence of the person can be determined, and data on the recent past (hours, days) can be obtained. In order to avoid the limitations experienced in blood substance analysis, analysis techniques by dried blood samples (DBS) have been developed. DBS is a very practical technique in terms of sampling, transportation, and storage of dried samples. In addition, it has advantages such as the long-term deterioration of the samples and the risk of haemolysis. This thesis, it is aimed to make a chromatographic determination of cannabis, amphetamine, 3,4-methylenedioxy-N-methylamphetamine (MDMA), morphine, and cocaine with DBS technique using different sorbents. In this context, it is aimed to investigate their suitability for the determination of illegal substances in the blood by developing nanofiber sorbents developed with the electrospinning technique in accordance with the working principle of DBS. Optimum chromatographic conditions of THC-COOH, amphetamine, MDMA, morphine and benzoylecgonine were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analytical method validation was performed using two different matrices for blood and dried blood samples (Whatman 903). With the electrospinning method, the fiber spinning solution was exposed to high voltage, polymers were electrically charged, and 5 different nanofibers containing cellulose and thermoplastic polyurethane (TPU) were synthesized. In the analyzes made with authentic blood samples and standard addition samples, studies were carried out for the selection of appropriate materials. 1 mL solvent was added to a 50 µL blood sample and liquid-liquid extraction was performed. The organic phase was transferred to a clean Eppendorf and evaporated under nitrogen to dryness. The residues were dissolved in 150 µL of mobile phase mixture and vials were injected to LC-MS/MS. DBS samples were dried at room temperature for 2 hours by dripping 50 µL of blood, and the dried spots were cut and transferred into 1 mL of extraction solvent. After 30 minutes of ultrasonic bath, the paper/sorbent was removed from the solvent and the procedure for blood sample was applied. Many different solvents and solvent mixtures were performed, and the most compatible results were obtained at the methanol-acetonitrile ratio of 40:60 (v/v). In the analytical methods developed, each analyte can be distinguished within the sample it contains, and the calibration curve correlation coefficient is >0.99 for each analyte. It is an accurate, precise, and reproducible validated method whereby each analyte is in the ±20% recovery range at low (25 ng/mL), medium (100 ng/mL), and high (200 ng/mL) concentrations and the correlation coefficient does not exceed 20%. The lower limit of quantification was for morphine 6.7, amphetamine 16.7, MDMA 22.7, benzoylecgonine 5.3, THC-COOH 25 ng/mL in blood, morphine 17.8, amphetamine 20.8, MDMA 22.7, benzoylecgonine 6.9, THC-COOH 25 ng/mL in DBS. In the stability study for 1, 10, and 30 days, each analyte was determined to be stable in blood and DBS. As a result of the comparison study, it was determined that the results of blood samples and DBS samples were compatible with each other (n=58, r=0.949), statistically. It has been determined that Whatman 903 filter paper gives the closest results with 48 hours-produced cellulose and cellulose-TPU nanofiber. Sorbents or cards used to dry blood samples are being developed and they are not yet produced for this purpose in our country. Preliminary studies have been completed for the development of a material supplied from abroad in our country for these developed nanofibers. A more effective, intended use, efficient product or products specific to analytes can be synthesized and create a new field in forensic toxicological studies.
Açıklama
Anahtar Kelimeler
Kan, Kurumuş Kan, Elektrospinning, Nanolif, Kromatografi, LC-MS/MS, Blood, Dried Blood, Electrospinning, Nanofiber, Chromatography, LC-MS/MS
