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EAB10008 Hypnotic Drugs Detection in Blood Sample

Application Introduction

This experiment used Qdaura® SPE-40 automated station to do the pre-treatment experiments of three different types of hypnotic drugs, barbiturates, tricyclic and benzodiazepine class, etc. and achieved relative ideal results.

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Introduction

This experiment used Qdaura® SPE-40 automated station to do the pre-treatment experiments of three different types of hypnotic drugs, barbiturates, tricyclic and benzodiazepine class, etc. and achieved relative ideal results.


Table 1 Hypnotics sample information

Sample   Name

Structure

Formula

Molecular   Weight

CAS   Number

Barbital

                          

C8H12N2O3

184.19

57

Phenobarbital

C12H12N2O3

232.24

50

Chlorpromazine

C17H19ClN2S

318.86

50

Clozapine

C18H19ClN4

326.82

5786

Estazolam

C16H11ClN4

294.74

29975

Aprobarbital

C10H14N2O3

210.23

77

Alprazolam

C17H13ClN4

308.76

28981

Experimental

Reagent material

Methanol, dichloromethane, ethanol, acetic acid were HPLC grade; disodium hydrogen phosphate, sodium dihydrogen phosphate were of analytical grade; ultra-pure water; blood;

Acetic acid - solution: Take 50 mL of deionized water with acetic acid adjusted to pH = 6;

Disodium hydrogen phosphate solution: 0.1 mol/L, weighed 35.81 g disodium hydrogen phosphate (Na2HPO4 • 12H2O), dissolved in water and dilute to 1 L;

Sodium dihydrogen phosphate solution: 0.1 mol/L, weighed 15.60 g disodium hydrogen phosphate (Na2HPO4 • 12H2O), dissolved in water and dilute to 1 L;

0.1 mol/L phosphate buffer solution (pH = 6): The 1.70 g of dibasic sodium phosphate and 12.14 g sodium dihydrogen phosphate was dissolved in 800 mL of deionized water, and then diluted with deionized water to 1 L, mix well. pH adjusted to 6.0 (with 0.1 mol/L sodium dihydrogen phosphate to lower pH; with 0.1 mol/L disodium hydrogen phosphate to increase the pH);

Hypnotic standard stock solution: mixed to standard 1 mg/mL, including barbiturates, phenobarbital, chlorpromazine, clozapine, estazolam, Appleton ratio properly, alprazolam, solvents for dry ethanol;

Hypnotic standard products working solution: take hypnotic standard stock solution 50 μL, diluted with ethanol to mix standard solution of 400 ppb;

 

Sample Preparation

Sample Extraction

Measured 2.0 mL blood sample, added 2 mL pH=6 phosphate buffer solution, vortex mixed 1min, then ultrasound 10 min, mixed and to be purified.

Sample purification

(1) Activation: 6 mL methanol and 6 mL of water were added into the Cleanert® PEP-2 cartridge in sequence.

(2) Sample Loading: 4 mL of the pre-treament plasma sample was load into the cartridge.

(3) Washing: 6 mL of acetic acid aqueous solution (pH = 6), discard the eluent. Suction the cartridge by pump for 10 min.

(4) Elution: Eluted the target compounds by 8mL of dichloromethane

Concentrated the elution to dryness by nitrogen and reconstituted it by 250 μL of ethanol, filtrated the solution by filter and detected by GC-MS.

Table 2 Qdaura SPE-40 automated extraction procedure

Step

Instruction

Solvent

Flow   rate

(mL/min)

Flow   volu

(mL)

1

Activation

Methanol

3

6

2

Activation

Water

3

6

3

Adding   sample

Air

2

10

4

Washing   sample tube

Acetic   acid - water

3

6

5

Washing   sample tube

Air

5

10

6

Washing   extraction column

Air

3

30

7

Collecting   an extraction column

Dichloromethane

2

8

8

Collecting   an extraction column

Air

3

10


Instrumentation

Chromatographic conditions

Instrumentation: GC-MS; Qdaura SPE-40 (automated solid phase extraction device)

HPLC Column: DA-5MS capillary column (30 m × 0.25 mm × 0.25 μm), the carrier gas is helium with the purity of 99.99%

Flow rate: 1 mL / min;

Initial column temperature: 130℃ , 10℃ /min heat up to 280℃ ,then keep 10 min; injector temperature is 270℃ .

MS conditions Ion source temperature: 230℃ , MS quadrupole temperature: 150℃ , the scan mode is SIM.

 

Result

(1) Chromatogram

 

Figure 1 Chromatogram of hypnotic drugs standard solution      

Figure 2 Chromatogram of blood sample

Figure 3 Chromatogram of hypnotic drugs spiked to blood sample

 

(2) Recovery data

Table 3 Recovery data of 7 kinds of hypnotic spiked in the plasma sample (400ng/mL)

Compounds

Retention   Time/min

Average   Recovery

RSD/%   (n=5)

Barbital

6.117

96.69

2.31

Phenobarbital

11.231

103.93

9.24

Chlorpromazine

16.050

73.76

5.37

Clozapine

20.207

90.91

6.33

Estazolam

20.751

98.85

10.57

Aprobarbital

7.521

95.62

8.90

Alprazolam

21.338

95.48

3.76

Conclusion

The experiment using Cleanert PEP-2 established pre-treatment methods of seven kinds of psychotropic drugs in blood, and combined Qdaura SPE-40 automated SPE station to measure the psychotropic drugs in blood samples. Experiments tested the amount of 400 ng/mL samples,and the results show that the recovery rate of this method is good, between 70% -110%, can be used to detect psychotropic drugs in the blood.

Order Information


Products

Specification

Cat.No

Cleanert® PEP-2

200 mg / 6 mL

PE2006-2

DA-5MS

30 m × 0.25   mm×0.25 μm

1525-3002

Qdaura® Automated SPE   Workstation

4 channels, 24   samples

SPE-40

1.5 mL vials

1.5 mL short   thread vial, amber glass, label and filling lines

AV1111-0

1.5 mL vials caps

9 mm screw neck   cap, center hole; red silicone/ white PTFE septa

AV2100-0

Filtration   membrane(Nylon)

φ13; 0.22 μm

AS021320-T

Disposable   Syringe

2   mL, needless

ZSQ-2ML

 

  • Cleanert PEP-2

    Cleanert PEP-2 is made of polydivinylbenzene on which the surface is functionalized with vinyl pyrrolidone and urea.

  • DA-5MS

    ● Identical selectivity to DB-5, but with much lower column bleed ● Best surface inertness for wide range of compounds ● Ideal for GC-MS analysis or new method developmentSimilar Phases: HP-5MS, DB-5MS, BPX-5MS, AT-5MS, Rxi-5MS, CP-Sil 8 CB

  • Qdaura Automated SPE System

    The Qdaura Automated SPE system is specifically designed for high throughput sample preparation. The system automates routine SPE procedures from conditioning, sample loading, washing, dry and elution. The Qdaura utilizes positive pressure to ensure more reproducible flow rates, more reproducible recoveries, less sample to sample contamination, and higher precision.

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