Antihistamin diekstraksi dari sampel darah utuh sebagai berikut: 30 mL larutan standar internal kerja
(10,00 mg mL 1 ) ditambahkan ke 1,0 mL sampel darah utuh berduri (LOQ, kalibrasi, dan QC) dan
casework; dengan demikian, konsentrasi masing-masing standar internal di semua sampel adalah
300,0 ng mL 1. Kemudian, volume 2 mL asetonitril ditambahkan ke semua sampel, di bawah
pencampuran pusaran, untuk presipitasi protein, dan sampel disentrifugasi pada 2000 rpm selama 5
menit. Supernatan dikumpulkan dan diuapkan hingga 1,0 mL di bawah aliran lembut N2 pada suhu
kamar. Kemudian, pH semua sampel disesuaikan menjadi 4,0 dengan penambahan 5 mL campuran
buffer asetat yang baru disiapkan (pH 4,0; 0,1 M): metanol (95: 5, v / v) dan sampel disentrifugasi
pada 2000 rpm selama 10 menit. Kolom SPE Bond Elut LRC Certify II dikondisikan dengan 3 mL
metanol dan 3 mL campuran buffer asetat yang baru disiapkan (pH 4,0; 0,1 M): metanol (95: 5, v / v)
sebelum pemuatan sampel. Aer pemuatan sampel, kolom kemudian dicuci dengan 3 mL air
deionisasi dan dikeringkan di bawah vakum tinggi $ 10 mm Hg selama 5 menit aer penambahan 70
mL aseton. Semua antihistamin, kecuali cetirizine, dielusi dua kali dengan 2 mL campuran dichlor
omethane yang baru disiapkan: isopropanol: amonium hidroksida (85: 15: 3, v / v / v). Kemudian,
kolom dicuci lagi dengan 1 mL air deionisasi dan dikeringkan di bawah vakum tinggi $ 10 mm Hg
selama 5 menit penambahan 70 mL n-heksana. Cetirizine dielusi dua kali dengan 2 mL campuran
dichlor omethane yang baru disiapkan : isopropanol : asam asetat glasial (85 : 15 : 3, v / v / v). Eluat
dikumpulkan dalam tabung bersih dan diuapkan hingga kering di bawah aliran lembut N2 pada suhu
40 C. Derivatisasi sampel yang diekstraksi dicapai dengan menambahkan 100 mL campuran anhidrida
asetat yang baru disiapkan: n-propanol (1: 1, v / v) dan inkubasi pada suhu 70 C selama 30 menit.
Aer pendinginan, semua larutan turunan evapo dinilai kekeringan di bawah aliran lembut N2 pada
suhu kamar dan sampel dipindahkan ke botol auto-sampler aer rekonstitusi dengan 70 mL etil
asetat. 1 mL disuntikkan ke dalam sistem GC-MS (mode splitless).
The precision and accuracy of the method were estimated as the relative standard deviation (% RSD)
and the percentage difference from the expected concentration (% Er), respectively. These
parameters were calculated by analyzing six (intra-day) and thirty (inter-day) spiked whole blood
samples at the three QC levels. The results for accuracy and precision at the three QC levels for all
analytes are presented in Table 3
The quantification of the selected NPS was performed using an Acquity UPLC I class system (Waters
Associates) for the chromatographic separation coupled to a triple quadrupole (Xevo TQ-S micro)
mass spectrometer equipped with an orthogonal Z-spray-electrospray interface (ESI) (Waters
Associates, Milford, MA, USA). Nitrogen was used as drying and nebulizing gas. The desolvation gas
flow was set to 1200 L/h and the cone gas flow to 50 L/h. A capillary voltage of 3 kV was used in
positive ionization mode. The nitrogen desolvation temperature was set to 600ºC and the source
temperature to 150ºC. Argon was used as collision gas and the injection volume was 10 µL.
The chromatographic separation was achieved at 55ºC using an Acquity UPLC BEH C18, (1.7 µm, 2.1 x
100 mm) (Waters Associates), at a flow rate of 300 µL/min. Mobile phase A was ammonium formate
1 mM with formic acid (0.01% v/v) in methanol. Mobile phase B was ammonium formate 1 mM with
formic acid (0.01% v/v) in water. A gradient program was used for the separation of the analytes; the
percentage of mobile phase A linearly changed as follows: 0 min, 15%; 1 min, 15%; 7.9 min, 95%; 8
min, 99%; 8.5 min, 99%; 9 min, 15%; 10 min, 15%. Analytes were determined by a Selected Reaction
Monitoring (SRM) method by acquiring two transitions for each compound. MassLynx software V4.1
and TargetLynx XS were used for data management.