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Microsoft word - z57010 tad 2 english rev.1.doc

TRICYCLIC ANTIDEPRESSANTS 2 in SERUM and/or in PLASMA by UV
CODE Z57010
(Doxepin, Amitriptyline and Nortriptyline )
INTRODUCTION

Tricyclic Antidepressants (abbreviation TCA) are a class of antidepressant drugs first used in the
1950s. They are named after the drug’s molecular structure, which contains three rings of atoms. They are used in numerous applications; mainly indicated for the treatment of clinical depression, neuropathic pain, nocturnal enuresis and attention-deficit hyperactivity disorder (ADHD), but they have also been used successfully for headache (including migraine headache), anxiety, insomnia, smoking cessation, bulimia nervosa, irritable bowel syndrome, narcolepsy, pathological crying or laughing, persistent hiccups, interstitial cystitis, ciguatera poisoning, and as an adjunct in For many years they were the first choice for pharmacological treatment of depression. Although still considered effective, they have been increasingly replaced by SSRIs and other newer drugs. Newer antidepressants are thought to have fewer side effects and are also thought to be less effective if used in a suicide attempt, as the treatment and lethal doses are farther apart than with the tricyclic antidepressants. They are sometimes still used to treat refractory depression that has failed to respond to standard SSRI therapy. The exact mechanism of action is not well understood, however it is generally thought that tricyclic antidepressants work by inhibiting the re-uptake of the neurotransmitters norepinephrine and serotonin by neurons. Tricyclic antidepressants may also possess any affinity for muscarinic and histamine H1 receptors to varying degrees, also increase the effects of H1 histamine, and thus Tricyclic antidepressants overdose is a significant cause of fatal drug poisoning. The severe morbidity and mortality associated with these drugs is well documented and due to their cardiovascular and neurological toxicity. Initial or mild symptoms include drowsiness, a dry mouth, nausea and vomiting. More severe complications, include hypotension, cardiac rhythm disturbances, hallucinations and seizures. EUREKA srl – LAB DIVISION
Head Quarter:
VAT N° 01547310423
Via E. Fermi 25
E-mail:info@eurekaone.com
60033 Chiaravalle (AN) ITALY
www.eurekaone.com
Tel. +39 071 7450790
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x + 39
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1 7496
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9
Release N° 001
November 2007
TECHNICAL FEATURES

Principle of the Method:

The Tricyclic Antidepressants are isolated purifying the serum/plasma with a clean-up column. After the
adding of a stabilization solution the eluate is directly injected into HPLC system.
Recovery :
Sensitivity :
Dynamic Range of the Method :
Therapeutic Range in serum :
Doxepin : 110 – 250 µg/l
Amitriptyline : 100 – 250 µg/l
Nortriptyline : 50 – 150 µg/l
Components of the kit :
All the reagents are ready-to-use and stable 3 years at 2–8 °C. The Calibrators are stable 24 months at 2-8 °C. Reagent A – Diluting Solution, 1 x 40 ml

Reagent B – Conditioning Solution N° 1, 1 x 300 ml

Reagent C – Conditioning Solution N° 2, 1 x 300 ml

Reagent D – Wash Solution N° 1, 1 x 300 ml

Reagent E – Wash Solution N° 2, 1 x 300 ml

Reagent F – Eluting Solution, 1 x 50 ml

Reagent G – Internal Standard Clomipramine Solution, 1 x 2 ml
See Warnings

Reagent H – Test Solution, 1 x 5 ml

Reagent P – Plasma Calibrator lyophil. for Doxepin, 1 x 5 ml
See Warnings

Reagent Q – Plasma Calibrator lyophil. for Amitriptyline and Nortriptyline, 1 x 5 ml See Warnings

Clean-up Columns, 100 Pcs

Reagent M – Mobile Phase, 5 x 500 ml
Minimum Instrumental equipment required:
Isocratic HPLC System with loop of 100 µl
Spectrophotometric Detector λ=216 nm
Chromatograms Recorder
Optional Equipment:
Whole Blood Collection Procedure :
Collect 5 ml of blood in a suitable test tube without gel or with anticoagulant EDTA. Centrifuge at 4000 rpm for 5 minutes. Separate the serum and store at – 20 °C. Stable 4 weeks. PREANALYTICAL PROCEDURE

Preparation of Test Solution at 1850 µg/l.

Pipette in a tube :
• 50 µl of Reagent H – Test Sol.
• 20 µl of Reagent G – Internal Standard Sol.

INJECTION :

• Inject 100 µl of this solution in the chromatographic system.
Verify that the Test Solution has retention time similar to fig. 1. If the Test is all right you can start with the
analytical procedure; if not, check the functionality of the analytical system.
Important : Don’t use this solution to calibrate!

ANALYTICAL PROCEDURE

STEP 1 : Preparation of Samples, Calibrator and Controls

Pipette in a tube :
Blank Sample
Calibrator
Water HPLC grade
Reagent P or Reagent Q – Calibrator
Reagent A – Diluting Sol.
Reagent G – Internal Standard Sol.
Agitare al vortex per 10 secondi
Vortex for 10 sec.

STEP 2 : Clean-up Columns Conditioning
Prepare a number of Clean-up columns like the number of samples to analyse + the Calibration Standard
• Condition clean up columns by fluxing 3 ml of Reagent B – Conditioning Sol. N° 1 and don’t let the
• Condition clean up columns by fluxing 3 ml of Reagent C – Conditioning Sol. N° 2 and don’t let the

STEP 3 : Samples Loading

Dispense 1,420 ml of sample, previously prepared, into Clean-up columns and discharge the percolated
liquid, if needed a vacuum system could be used for percolation, at speed of 1 ml / min ( drop by drop ).


STEP 4 : Washing
Wash with 3 ml of Reagent D – Washing Sol. N° 1 Let dry with a light flux of air and discharge the
percolated liquid
Wash with 0,8 ml of Reagent E – Wash Sol. N° 2 Let dry with a light flux of air and discharge the
percolated liquid

STEP 5 : Elution
Elute with 0,5 ml of Reagent F – Eluting Sol. Let dry with a light flux of air and discharge the
percolated liquid
• Dilute the eluate with 0,4 ml of H2O HPLC grade Vortex for 10 sec.
N.B.: at this step, the sample is stable 1 day at 2-8 °C

INJECTION :
• Inject 100 µl of the eluate into the chromatographic system. TRICYCLIC ANTIDEPRESSANTS 2 - Warnings

REAGENT G : INTERNAL STANDARD CLOMIPRAMINE SOLUTION

The consumption of Clomipramine, contemporaneous at TAD2, determinates a wrong
quantitative determination.

REAGENT H : TEST SOLUTION

TRICYCLIC ANTIDEPRESSANTS 2 :
Amitriptyline
Nortriptyline

REAGENT P : PLASMA CALIBRATOR LYOPHIL. - Lot.N°9226 (Doxepin)
TRICYCLIC ANTIDEPRESSANTS 2 :
Use and Reconstitution: Urine Calibrators are used for calibration of the
HPLC system. This lyophilised calibrator has to be prepared like a patient
sample. Add exactly 5.0 ml of HPLC water to the vial and mix for 15 min.
When all material is dissolved, the solution is ready to use.
Stability: 24 months if stored at 2-8 °C. After reconstitution the stability of
the analytes is at least 25 days when stored at 2–8 °C. Don’t use after
expiry date.
Packaging : 1 x 5 ml
Warning: The calibrator derives from human plasma, so it could be
potentially infected. It must be handled with care.

REAGENT Q : PLASMA CALIBRATOR LYOPHIL. - Lot.N°9224 (Amitriptyline,Nortriptyline)
TRICYCLIC ANTIDEPRESSANTS 2 :
Amitriptyline
Nortriptyline
Use and Reconstitution: Urine Calibrators are used for calibration of the
HPLC system. This lyophilised calibrator has to be prepared like a patient
sample. Add exactly 5.0 ml of HPLC water to the vial and mix for 15 min.
When all material is dissolved, the solution is ready to use.
Stability: 24 months if stored at 2-8 °C. After reconstitution the stability of
the analytes is at least 30 days when stored at 2–8 °C. Don’t use after
expiry date.
Packaging : 1 x 5 ml
Warning: The calibrator derives from human plasma, so it could be
potentially infected. It must be handled with care.

SPECTROPHOTOMETRIC DETECTOR PARAMETERS

INTEGRATION TIME

HPLC COLUMN PROTECTION

To save the analytical column Reverse Phase GENESIS 4,6 x 150 mm, 4 µ, the use of PRECOLUMN CARTRIDGE, cod. ZPHKJO4282 and of PRECOLUMN PREFILTERS (1 x 10 pcs.) cod. ZPHAJO4287 is obligatory. HPLC COLUMN CONDITIONING

Install a new analytical column Synergi MAX RP 4,6 x 150 mm, 4 µ. Disconnect the detector and filter 30 ml of H2O: Methanol ( 20 : 80 v/v ) solution and subsequently 30 ml of H2O per HPLC, set flow at 1,2 ml / min. Don’t recycle the washing solutions. Filter the mobile phase
with a vacuum system and a suitable filter of 0,22 µ. Condition the column with the mobile
phase at a flow of 1,2 ml / min. and discharge the first 30 ml. Condition further on the column for
30 min. also at recycling phase. Finally inject the Chemical Standard and verify the quality of the
HPLC run.
It is NOT possible to make analysis at recycling phase.
If room temperature is > 20 °C store the Mobile Phase at 2-8 °C between an analytical session and another.
COLUMN CLEANING

Disconnect the detector. Flux 30 ml of H2O and discharge. Flux a solution made of H2O : Methanol or Acetonitrile (20 : 80 v/v) for 30 min and discharge. When you re-use the column, flux 30 ml of H2O before a new conditioning with the Mobile Phase.
HPLC PARAMETERS

PRESSURE

INTEGRATOR HP – 3394 / 3395 / 3396 PARAMETERS

ATTENUATION

OPERATIONAL COMPUTER PARAMETERS

IN CONFORMITY WITH THE SPECIFICATION OF OPERATIONAL COMPUTER SOFTWARE
TRICYCLIC ANTIDEPRESSANTS 2 IN SERUM and/or IN PLASMA Plasma Calibrator lyophil.
Fig. 2
Plasma Calibrator lyophil.
R.T. 18.92 Internal Standard Clomipramine R.T. 18.96 Internal Standard Clomipramine TRICYCLIC ANTIDEPRESSANTS 2 IN SERUM and/or IN PLASMA Plasma Control lyophil.
Plasma Control lyophil.
R.T. 19.10 Internal Standard Clomipramine R.T. 18.88 Internal Standard Clomipramine TRICYCLIC ANTIDEPRESSANTS 2 IN SERUM and/or IN PLASMA Enriched sample high
Fig. 6
Enriched sample low
R.T. 19.06 Internal Standard Clomipramine R.T. 19.14 Internal Standard Clomipramine

Source: http://www.rotakim.com/ckfinder/userfiles/files/tca2.pdf