Alex Birsan (Presenter)
Phytronix Technologies
Authorship: Alex Birsan, Pierre Picard, Serge Auger, Annie-Claude Bolduc and Jean Lacoursière
Phytronix Technologies Inc., 4535 Boulevard Wilfrid-Hamel, Suite 120, Quebec, Quebec, Canada G1P 2J7
| Short Abstract According to a 2011 National Center for Health Statistics (NCHS) report, the rate of Antidepressants use in USA increased by almost 400% between 2005 and 2008 for people older than 12 years old. The federal government’s health statisticians figure that about one in every 10 Americans takes at least one antidepressant. By their calculation, antidepressants were the third most common prescribed medication taken by Americans. These numbers are only likely to increase over the years. Using mass spectrometry combined with high-throughput LDTD ion source enhances specificity at equivalent or better speed for the quantification of 7 Antidepressants in human urine. Using only a basic liquid-liquid extraction for the sample preparation, we are able to achieve a robust method with precision and accuracy at a speed of 9 seconds per sample. |
Long Abstract
Overview
According to a 2011 National Center for Health Statistics (NCHS) report (1) , the rate of Antidepressants use in USA increased by almost 400% between 2005 and 2008 for people older than 12 years old. The federal government’s health statisticians figure that about one in every 10 Americans takes at least one antidepressant. By their reckoning, antidepressants were the third most common prescribed medication taken by Americans. These numbers are only likely to increase over the years. Using mass spectrometry combined with high-throughput LDTD ion source enhances specificity at equivalent or better speed for the quantification of 7 Antidepressants in human urine. Doing only a basic liquid-liquid extraction for the sample preparation, we are able to achieve a robust method in precision and accuracy at a speed of 9 seconds per sample.
LDTD Ionization Source
The LDTD uses a Laser Diode to produce and control heat on the sample support which is a 96 well plate. The energy is then transferred through the sample holder to the dry sample which vaporizes prior to being carried by a gas in an APCI region. High efficiency protonation with strong resistance to ionic suppression characterize the ionization due to the absence of solvent and mobile phase. This allows very high throughput capabilities of 6 seconds sample-to-sample analysis time, without any carry over.
Sample preparation
Glucuronide hydrolysis
• 50 µL patient sample (or standard)
• 2.5 µL IS (Clomipramine-d3 10 µg/mL in MeOH:Water/1:1)
• 10 µL Purified β-glucuronidase (IMCSzyme™)
• 12.5 µL Hydrolysis buffer (IMCSzyme™)
• Cap and mix
• Incubation at 55°C for 30 minutes
Basic Liquid-Liquid Extraction
• 100 µL Na2CO3 buffer (0.5M pH 10)
• 800 µL Hexane:EtAc / 25:75
• Vortex
• Wait for phase separation
• Transfer 5 µL of organic upper layer in a LazWell™ plate
• Dry prior to analysis
Instruments setting
1) Mass spectrometer:
Mass spectrometer AB Sciex 5500 QTrap®
MRM transitions used with 3 µA corona discharge current, 5 msec dwell time, and an 80 Volts DP
Compound Q1 Q3 CE(V)
Amitriptyline (Quant) 278 233 25
Amitriptyline (Conf) 278 117 30
Clomipramine (Quant) 315 86 25
Clomipramine (Conf.) 315 58 55
Cyclobenzaprine (Quant) 276 215 50
Cyclobenzaprine (Conf) 276 231 25
Desipramine (Quant) 267 72 20
Desipramine (Conf) 267 44 55
Doxepin (Quant) 280 117 30
Doxepin (Conf) 280 235 25
Imipramine (Quant) 281 86 20
Imipramine (Conf) 281 236 25
Nortriptyline (Quant) 264 233 20
Nortriptyline (Conf) 264 117 30
Clomipramine-d3 320 61 55
2) LDTD:
LDTD model S-960 operated with a gas flow rate of 3 L/min and a laser pattern ramp from 0 to 65% in 6 seconds and maintaining this power level for 1 second.
Methodology
The standard calibration curve is made in blank urine. A calibration curve ranging from 15.6 to 2000 ng/mL is used. Low, medium and high QC levels are prepared. These QCs are used for intra-run and inter-run bias and precision measurements. The internal standard is prepared with a stock solution of Clomipramine-d3, spiked in methanol:water (1:1) for a final concentration of 10 µg/mL. Each sample is treated with β-glucuronidase to obtain free drug, followed with a basic liquid-liquid extraction. All samples are analyzed using LDTD-MS/MS method.
Results
Linearity from the calibration curve is expressed by the correlation coefficient R presented in the following table. All curves have R coefficients of 0.995 or better for LDTD-MSMS analysis.
Table 1: Bias / Precision intra-Assay for clomipramine in urine matrix
LLOQ QC-Low QC-Med QC-High ULOQ
Conc. (ng/mL) 15.6 62.5 250 1000 2000
N 6 6 6 6 6
Mean (ng/mL) 16.1 63.6 253.3 1027.3 1971.5
%CV 10.7 5.9 7.1 4.6 4.8
%Bias 3.1 1.7 1.3 2.7 -1.4
Table 2: Bias / Precision inter-Assay for clomipramine in urine
QC-Low QC-Med QC-High
Conc. (ng/mL) 62.5 250 1000
N 24 24 24
Mean (ng/mL) 64.3 246.8 1005.1
%CV 6.1 4.9 4.0
%Bias 102.8 98.7 100.5
All QCs and calibration standards have bias value within the acceptance criteria (±20%) and a precision values ≤ 15%, as required in the method validation for intra-run and inter-run bias and precision. Ionization suppression/enhancement evaluation was performed by spiking ten different matrixes at low level QC. Bias and precision criteria were applied. The carry-over is evaluated by the analysis of three blanks after the highest standard. The blank peak areas were evaluated against the mean peak area of the lower standard to determinate the interference percentage. Interference evaluation of blank matrix, internal standard stable isotope and other commonly encountered analytes were evaluated. No interferences were noted, with a total of 35 drugs (benzodiazepines, barbiturates, amphetamines, cannabinoids, etc) spiked in blank urine. No ionization suppression was observed, as the lowest standard was spiked in 10 different urine matrices. To check the stability of the samples in their wet state, the extraction were kept at 4 °C and analyzed after 24 hours. All bias and precision results were within the acceptable range. To determine the stability of the samples in their dry state, all the QCs and standard were spotted. The plate was then kept at room temperature (22°C) and analyzed after 24 hours. All bias and precision results were within the acceptable range.
Conclusion:
Method Validation of TCA drugs in urine using β-glucuronidase hydrolysis and basic liquid-liquid extraction with subsequent analysis by LDTD-MS/MS at 9 seconds per sample was achieved. All acceptance criteria parameters were attained for the method validation.
The LDTD technology combined with a mass spectrometer system allows ultra-fast and specific TCA drugs quantification in urine samples with minimal sample preparation.
References & Acknowledgements:
Pratt LA, Brody DJ, Gu Q. Antidepressant use in persons aged 12 and over: United States, 2005–2008. NCHS data brief, no 76. Hyattsville, MD: National Center for Health Statistics. 2011.
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Phytronix Technologies |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |