Since the hair root is vascularized during its growth, illicit drugs present in the blood stream may enter the hair shaft via the root where they will be sequestered. Therefore, the use of illicit drugs can be revealed by analyzing a small hair sample. To increase the analysis throughput of hair samples, the Luxon Ion Source® coupled to tandem mass spectrometry (MS/MS) was used for the identification and quantification of drugs of abuse.

Our goal for this application note is to use an automated sample preparation method for the screen of drugs in hair sample using a single operation in LUXON-MS/MS.

LUXON-MS/MS offers specificity combined with an ultra-fast analysis for an unrivaled screening method. To develop this application, we focused on performing a quick and simple sample preparation. Drugs are analyzed simultaneously with quantitative screening results obtained in less than 9 seconds per sample.

Luxon Ionization Source

The Luxon Ion Source® (Figure 1) is the second-generation sample introduction and ionization source based on the LDTD® technology for mass spectrometry. Luxon Ion Source® uses Fiber-Coupled Laser Diode (Figure 2) to obtain unmatchable thermal uniformity providing more precision, accuracy and speed. The process begins with dry samples which are rapidly evaporated using indirect heat. The thermally desorbed neutral molecules are carried into a corona discharge region. High efficiency protonation and strong resistance to ionic suppression characterize this type of ionization and is the result of the absence of solvent and mobile phase. This thermal desorption process yields high-intensity molecular ion signal in less than 1 second sample-to-sample and allows working with very small volumes.

Figure 1 – Luxon Ion Source®

Figure 2 – Schematic of the Luxon Ionization Source

Sample Preparation Method

Automated Sample Extraction

• 10 mg of hair cut into small pieces are transferred into barcoded tubes, readable by the Azeo extraction system.
• A pre-wash of the hair is performed to remove external contaminants using 1 mL of Methanol, soak 5 minutes and decanted.
• 1 mL of methanol containing TFA at 0.5% (with internal standard) is added and samples are soaked at 60 degrees Celsius for 1h45. Samples are then sonicated for 15 minutes.
• After the extraction, vials were transferred to the Azeo system.
• Each barcoded vial was scanned by the Azeo Liquid Handler system (Figure 3).
• 20 µL of desorption solution (KH₂PO₄ (0.1 mM) in water) were mixed with 20 µL of sample.
• 8 µL of mixture were deposited onto a 96-LazWell plate.
• Samples were evaporated to dryness at 40 degrees Celsius for 8 minutes.
• Luxon-MS/MS analysis is performed after a complete evaporation.

Figure 3 – Automated extraction system

LDTD®-MS/MS Parameters

LDTD

Model: Luxon SH-960, Phytronix

Carrier gas: 6 L/min (air with 20 µL/min water)

Laser pattern:

• 3-second ramp to 55% power
• Hold 2 seconds at 55% power

MS/MS

MS model: LC-8060, Shimadzu

Ionization: APCI

Results and Discussion

Initial Cut-off Test

Table 3 shows the suggested screening cut-offs currently used in the industry.

Desorption peak

Figure 4 shows a typical desorption peak for a blank sample for Cocaine and Figure 5 shows a typical desorption peak for a cut-off sample for Cocaine. Similar results were obtained for the other drugs.

Figure 4 – Desorption peak of blank sample for Cocaine

Figure 5 – Desorption peak of cut-off sample for Cocaine

Precision

Spiked samples around the decision point and blank solutions are used to validate the precision of the method. The peak area against the internal standard (IS) ratio was used to normalize the signal. Replicate extractions are deposited on a LazWell plate and dried before analysis.

The following acceptance criteria were used:

• Each concentration must not exceed 20% CV.
• Mean concentration ± 2 times the standard deviation must not overlap with other concentrations at the cut-off.

Table 4 shows the precision results at the cut-off level.

Conclusion

Luxon Ion Source® combined to a Shimadzu LC-8060 mass spectrometer system allows ultra-fast (9 seconds per sample) screening of drugs in hair using a simple and automated sample preparation method.

The post Screening in Hair for Drugs (On Shimadzu Model) appeared first on Phytronix.

The US Department of Health and Human Services (via the SAMHSA agency) has established scientific and technical guidelines for federal workplace programs of drug testing in urine.

Our goal for this application note is to use an automated sample preparation method for a drug panel in urine using a single operation in LUXON-MS/MS.

LUXON-MS/MS offers specificity combined with an ultra-fast analysis for an unrivaled screening method. To develop this application, we focused on performing a quick and simple sample preparation. Fourteen drugs are analyzed simultaneously with quantitative screening results obtained in less than 8 seconds per sample. Each drug has been screened based on the SAMHSA guidelines cut-offs.

Luxon Ionization Source

The Luxon Ion Source® (Figure 1) is the second-generation sample introduction and ionization source based on the LDTD® technology for mass spectrometry. Luxon Ion Source® uses Fiber-Coupled Laser Diode (Figure 2) to obtain unmatchable thermal uniformity providing more precision, accuracy and speed. The process begins with dry samples which are rapidly evaporated using indirect heat. The thermally desorbed neutral molecules are carried into a corona discharge region. High efficiency protonation and strong resistance to ionic suppression characterize this type of ionization and is the result of the absence of solvent and mobile phase. This thermal desorption process yields high-intensity molecular ion signal in less than 1 second sample-to-sample and allows working with very small volumes.

Figure 1 – Luxon Ion Source®

Figure 2 – Schematic of the Luxon Ionization Source

Sample Preparation Method

Sample Collection

Urine samples were collected and transferred into barcoded tubes, readable by the Azeo extraction system.

Automated Sample Extraction

Each barcoded vial was scanned by the Azeo liquid handler and an automatic batch file was created. The Azeo extraction system (Figure 3) is used to extract the samples using the following conditions:

• 33.8 µL of Internal standard were added to each sample
• 112.5 µL of urine sample were transferred from the vials to a deep-well plate placed in the Lumo Vortexer
  • Mix
• 33.8 µL b-Glucuronidase-RT Enzyme/Hydrolysis buffer were added to each sample
  • Mix and incubate at room temperature for 15 minutes
• 225 µL Extraction buffer and 450 µL Acetonitrile were added into the deep-well plate
  • Mix and centrifuge 3 minutes/3500 rpm for phase separation
• Mix 65 µL of desorption buffer with 100 µL of upper layer
• Spot 6 µL of the mixture onto a LazWell96 plate
  • Dry 6 minutes at 40°C

Figure 3 – Automated extraction system

LDTD®-MS/MS Parameters

LDTD

Model: Luxon S-960, Phytronix

Carrier gas: 6 L/min (air)

Laser pattern:

• 3-second ramp to 65% power
• Hold 2 seconds at 65% power

MS/MS

MS model: Q-Trap System® 5500, Sciex

Scan Time: 5 msec

Total run time: 8 seconds per sample

Ionization: APCI

Analysis Method: MRM mode

Results and Discussion

Initial Cut-off Test (ng/mL)

A drug list and screening cut-off suggested by the SAMHSA guidelines can be found in Table 3.

Precision

Spiked samples around the decision point (50% cut-off: QC-L, cut-off: CO and 200% cut-off: QC-H) and blank solutions are used to validate the precision of the method. The peak area against the internal standard (IS) ratio was used to normalize the signal. Replicate extractions are deposited onto a LazWell plate and dried before analysis.

The following acceptance criteria were used:

• Each concentration must not exceed 20% CV
• The mean concentration ± 2 times the standard deviation must not overlap with other concentrations at the cut-off.

For the inter-run precision experiment, each fortified sample set is analyzed in triplicate on five different days. Table 4 shows the inter-run precision results. No overlapping at the cut-off is observed for BZE, a cocaine metabolite, and the %CV was below 20%. Similar results are obtained for the other drugs in the panel.

For the intra-run precision experiment, each fortified sample is extracted and analyzed in 8 replicates. Area ratio results are plotted using the ± 2 STD error bars. Figure 4 shows the intra-run results for BZE. No overlapping is observed for each concentration and the %CV was below 20%. Similar results are obtained for the other drugs in the panel.

Figure 4 – Intra-Run Precision Curves for BZE

Multi-matrix evaluation

Urine samples were collected from ten different volunteers. Samples are screened to verify the presence of each analyte (all samples showed negative results). To study the matrix effect, the different drugs are spiked at 50% cut-off (QC-L) and 200% cut-off (QC-H) and screened as unknown. Table 5 shows the screening result of three of them. Samples are spiked at QC-L and QC-H are detected as negative and positive, respectively.

Wet Stability of Sample Extracts

Following the extraction, sample extracts are kept at 4°C in closed containers. After 3 days, sample extracts are spotted on a LazWell plate, dried and analyzed. Precision at 50% cut-off standard is reported in Table 6 for BZE. All the results are within the acceptable range (criteria %CV ≤20%) for 3 days at 4°C. Similar results are obtained for the other drugs.

Dry Stability of Samples Spotted in LazWell

Extracted samples are spotted onto a LazWell plate and kept at room temperature before analysis. Precision at 50% cut-off standard is reported in Table 6 for BZE. All the results are within the acceptable range (criteria %CV ≤20%) for 30 minutes at room temperature. Similar results are obtained for the other drugs.

Conclusion

Luxon Ion Source® combined with Sciex Q-Trap 5500 mass spectrometer system allows ultra-fast (8 seconds per sample) screening of SAMHSA drug panel in urine using a simple and automated sample preparation method.

The post SAMHSA Drug Panel Screening in Urine appeared first on Phytronix.

Over the last decade, plant-based products laced with synthetic cannabinoids have increased. The analysis of the synthetic cannabinoid content in plant material is needed for health hazard assessments. Different structural classes (dibenzopyrans, cyclohexylphenols, naphthoylindoles, benzoylindoles, phenylacetylindoles, tetramethylcyclopropylindoles) are used as synthetic cannabinoids.

For this project, we propose to perform a serial addition of synthetic cannabinoids on plant material and define a screening extraction method using the LUXON coupled to a mass spectrometer (LUXON-MS/MS) as a fast-analytical technique.

LUXON ionization source

The Luxon Ion Source® (Figure 1) is the second-generation sample introduction and ionization source based on the LDTD® technology for mass spectrometry. Luxon Ion Source® uses Fiber-Coupled Laser Diode (Figure 2) to obtain unmatchable thermal uniformity providing more precision, accuracy and speed. The process begins with dry samples which are rapidly evaporated using indirect heat. The thermally desorbed neutral molecules are carried into a corona discharge region. High efficiency protonation and strong resistance to ionic suppression characterize this type of ionization and is the result of the absence of solvent and mobile phase. This thermal desorption process yields high-intensity molecular ion signal in less than 1 second sample-to-sample and allows working with very small volumes.

Figure 1 – Luxon Ion Source®

Figure 2 – Schematic of the Luxon Ionization Source

Sample Preparation Method

For the screening method, the following synthetic cannabinoids are used: JWH-018, AM-2201, 5-Fluoro PB-22, MAM-2201, XLR-11, AB-FUBINACA, APINACA and PB-22. 40 mg of plant material are weighed in a glass tube. 10 µL of a spiking solution (400, 200, 40, 20 and 4 µg/mL synthetic cannabinoids mixture) are added to get 100, 50, 10, 5 and 1 µg/g concentrations, respectively. The solvent is evaporated from the plant material before the extraction for the LDTD-MS/MS analysis.

Synthetic cannabinoids are sprayed on the surface of the leaf, so no attempt was made to homogenize the plant material content prior to sampling and analysis. Extraction is carried out as flows for spiked plant materials. 40 mL of ter-Butyl-Methyl ether containing internal standard (THC-d3 at 100 ng/mL) are added on spike sample. Tubes are capped, vortexed, then sonicated for 5 minutes. After centrifugation (5000 rpm, 2 minutes), 4 µL of the solution are spotted into LazWell96 plates and evaporated to dryness. LDTD-MS/MS analysis is done after a complete evaporation.

LDTD-MS/MS Parameters

LDTD

Model: Phytronix, LUXON SH-960

Carrier gas: 3 L/min (air)

Laser pattern: 6-second ramp to 50% power

MS/MS

Model: Shimadzu LCMS-8060

Ionization: APCI

Positive MRM transition

Note: Hydroxy metabolites are used for the experiment. Restricted compounds are not available.

Results and Discussion

Linearity

Synthetic cannabinoids spiked set at the following calibration range are extracted: 1 to 100 µg/g. Correlation coefficients are equal or greater than 0.99 for the quantification curve of all synthetic cannabinoids. Figure 3 shows the calibration curve of PB-22. Figure 4 shows the typical blank and PB-22 (1 µg/g) desorption peaks with a 0.16-minute window.

Figure 3 – PB-22 calibration curve

A) Blank

B) Standard : 1µg/g

Figure 4 – Blank (A) and PB-22 (B) desorption peaks

Synthetic cannabinoids recovery

A spiked sample (10 µg/g) and blank are extracted. Blank extract is then spiked and used as a 100% recovery reference. The concentration of the reference and the spiked sample are evaluated against the calibration curve. A recovery between 86.0 and 108.3 % is obtained. Results are shown in Table 2.

Precision around lower limit of detection (LOD)

Spiked samples around the lower limit of detection (LOD) and blank solutions are used to validate the precision of the method. Each concentration must not exceed 20% CV and the mean concentration ± 2 times the standard deviation (± 2 SD) must not overlap with other concentrations at the decision point. The peak area against IS ratio was used to normalize the signal. Replicate extractions are deposited on a LazWell plate and dried before analysis. No overlapping at the LOD is observed for all curves and the CV% was below 15%. Results using the ± 2 SD overlay are plotted. Figure 5 shows the results.

(A)

(B)

(C)

(D)

(E)

(F)

(G)

(H)

Figure 5 – 2 SD overlay plot

Conclusion

LUXON technology combined with an LCMS-8060 system allows ultra-fast (8 seconds per sample) and precise screening of synthetic cannabinoids on plant material using a simple plant dilution with internal standards.

The post Analysis of Synthetic Cannabinoids in Plant Materials appeared first on Phytronix.

Phosphatidylcholines (PCs) are the major constituents of cell membranes. They are used as biomarkers for several diseases. This glycerophospholipid group contains a polar phosphocholine head connected to fatty acid sidechains via a glycerol backbone. Varying lengths and saturation of fatty acids may be observed. The fatty acids are connected via two types of chemical bounds on a glycerol backbone: ester (acryl: a) or ether (e). The following nomenclature will be used:

Ex: PC_ae C32:1

• PC: Phosphatidylcholine head
• ae: Fatty acid chemical bonds: acryl (a) or ether (e)
• C32: Sum of Carbons on both fatty acid chains
• 1: Number of double bonds.

Our goal for this application note is to present a generic method that allows the quantitation of various phosphatidylcholine (PCaa, PCae and LPCa) in plasma in less than 8 seconds per sample. The automated sample preparation is reduced to a minimum to keep up with the analysis throughput.

Luxon Ionization Source

The Luxon Ion Source® (Figure 1) is the second-generation sample introduction and ionization source based on the LDTD® technology for mass spectrometry. Luxon Ion Source® uses Fiber-Coupled Laser Diode (Figure 2) to obtain unmatchable thermal uniformity giving more precision, accuracy and speed. The process begins with dry samples which are rapidly evaporated using indirect heat. The thermally desorbed neutral molecules are carried into a corona discharge region. High-efficiency protonation and strong resistance to ionic suppression characterize this type of ionization and it is the result of the absence of solvent and mobile phase. This thermal desorption process yields high-intensity molecular ion signal in less than 1 second sample-to-sample and allows working with very small volumes.

Figure 1 – Luxon Ion Source®

Figure 2 – Schematic of the Luxon Ionization Source

Sample Preparation Method (Automated)

An automated system (Figure 3) is used to extract the samples using the following conditions:

• 10 µL of plasma sample
• 10 µL of internal standard at 12 µM in IPA:ACN:Water (75:20:5)
• 150 µL of Methanol
  • Vortex
• 500 µL of MTBE
  • Vortex
• 150 µL of Water
  • Vortex and phase separation by gravity
• Spot 5 µL of the upper-layer phase on a coated LazWell96 plate
  • Dry 2 minutes at room temperature with air flow
• Add 5 µL of the desorption solution
  • Evaporate to dryness

Figure 3 – Automated extraction system

Luxon-MS/MS Parameters

Luxon

Model: Luxon S-960, Phytronix

Carrier gas: 6 L/min (air)

Laser pattern:

• 3-second ramp to 100% power
• 3-second hold at 100% power

MS/MS

MS model: Q-Trap System® 5500, Sciex

Scan Time: 5 msec

CE: 30

Total run time: 8 seconds per sample

Ionization: APCI (positive)

Analysis Method: MRM mode

A common loss of 184 amu associated to the polar phosphocholine head is used to generate the MRM method. Some examples of the MRM transitions used can be found in Table 1.

Results and Discussion

Phospholipids analysis

This method evaluates the phospholipid profile based on the sum of carbon, hydrogen, and oxygen atoms on the fatty acid sidechain. Also, the peak area corresponds to the sum of the potential isobaric phosphatidylcholine according to the following rules:

Precision

For the intra-run precision experiments, five different samples are analyzed in six replicates. Table 3, Table 4 and Table 5 show the intra-run results for PC_aa C34:1, PC_aa C34:2 and PC_aa C36:2, respectively. No sample exceeds 15% CV. Similar results are obtained for the phospholipid transitions.

To determine the inter-run precision, five different samples are analyzed in three runs. Table 6, Table 7 and Table 8 show the inter-run results for PC_aa C34:1, PC_aa C34:2 and PC_aa C36:2, respectively. No sample exceeds 15% CV. Similar results are obtained for the phospholipid transitions.

Method Comparison Evaluation

Plasma samples from healthy patients (N=24) have been tested with this method to correlate with results obtained with the assay reference kit AbsoluteIDQ (N=220). The median concentration values were determined for 37 different phosphatidylcholines and used for the statistical evaluation. Table 9 shows the phosphatidylcholines used for the method comparison.

The Passing-Bablok regression (Figure 4) reveals a correlation and no significant deviation from linearity. The Bland and Altman plot (Figure 5) shows the mean value bias of the two methods. All samples are within the confidence interval of 95%.

Figure 4 – Passing-Bablok regression curve

Figure 5 – Bland and Altman plot

Conclusion

A system combining Luxon Ion Source® with a Sciex 5500 Q-Trap mass spectrometer allows the ultra-fast (8 seconds per sample) phosphatidylcholine profiling in plasma samples using a simple and automated sample preparation method.

The post Phosphatidylcholine Profile Evaluation in Human Plasma appeared first on Phytronix.

Hydrocarbons C20 to C80 are usually analyzed by GC-MS systems using the Electron Ionization (EI) mode since LC-MS does not properly ionize this type of molecule. To increase the sample throughput, the Laser Diode Thermal Desorption (LDTD) technology shows promising results for the ionization and analysis of this molecular group. Luxon combined with a High-Resolution Mass Spectrometer (HRMS) is used to analyze hydrocarbon chains from commercial oil. The Luxon-HRMS is a rapid analysis approach in which molecules are thermally desorbed and then are channeled, using a carrier gas, to a corona discharge region for ionization prior to detection via a mass spectrometer. C20, C26, C40 and C50 straight-chain alkanes are used to validate the ionization process.

We propose to perform a quick hydrocarbon profile evaluation using Laser Diode Thermal Desorption High Resolution Mass Spectrometry (LDTD-HRMS).

Luxon Ionization Source

The Luxon Ion Source® (Figure 1) is the second-generation sample introduction and ionization source based on the LDTD® technology for mass spectrometry. Luxon Ion Source® uses Fiber-Coupled Laser Diode (Figure 2) to obtain unmatchable thermal uniformity providing more precision, accuracy and speed. The process begins with dry samples which are rapidly evaporated using indirect heat. The thermally desorbed neutral molecules are carried into a corona discharge region. High efficiency protonation and strong resistance to ionic suppression characterize this type of ionization and is the result of the absence of solvent and mobile phase. This thermal desorption process yields high-intensity molecular ion signal in less than 1 second sample-to-sample and allows working with very small volumes.

Figure 1 – Luxon Ion Source®

Figure 2 – Schematic of the Luxon Ionization Source

Sample Preparation Method

Standard

2 µL of Eicosane (1.3 mg/mL), Hexacosane (1.4 mg/mL), Tetracontane (0.65 mg/mL) and Pentacontane (0.15 mg/mL) in toluene are spotted into 96-LazWell plates and evaporated to complete dryness. Luxon-HRMS analysis is done after a complete evaporation.

Sample

2 µL of commercial oil is diluted in toluene at 1 ppm, spotted into 96-LazWell plates and evaporated to complete dryness. Luxon-HRMS analysis is done after a complete evaporation.

LDTD-HRMS Parameters

LDTD

Model: Phytronix, T-960

Carrier gas: 3 L/min (air)

Laser pattern: 3 second ramp to 45% power

MS/MS

Model: Q-Exactive, Thermo Scientific

Ionization: APCI (Positive)

Scan mode: 200 to 1200 m/z

Results and Discussion

Hydrocarbon Ionization process

During the desorption process, a dehydrogenation occurs on the LazWell plate surface to generate a double bond. The alkene formed is transferred via the carrier gas and ionized by the corona discharge. Figure 3 shows the dehydrogenation process.

Figure 3 – Dehydrogenation process standard analysis

Five successive wells are desorbed in the same MS file (Blank, C20, C26, C40 and C50). The mass spectrum and exact mass extraction are acquired. Results are reported in Table 1 and in Figure 4.

Figure 4 – C20 standard desorption

Oil sample profile analysis

Commercial oil is diluted with toluene at 1 ppm and analyzed by Luxon-HRMS. The Figure 5 shows the exact mass profile. The analysis of the High-Resolution mass spectrum highlighted two different profile distributions.

To evaluate the profile structures, the exact mass is extracted, and peak areas are reported. The major profile (Profile 1) is composed of the highest hydrocarbon peaks of the same unsaturation number (6) from all the sub-profile distributions. Profile 2 shows the unsaturation distribution of molecules having 27 carbon atoms. A similar unsaturation distribution is observed for all alkenes (C20, C21, C22, …).

Figure 5 – Commercial oil exact mass profile

Profile 1 – Hydrocarbon distribution of 6 degrees of unsaturation

Using the accurate mass spectra acquired and an elemental composition tool, molecules having an unsaturation degree of 6 are reported. Figure 6 shows the peak area profile of this group. Table 2 shows the empirical formula and exact mass.

Figure 6 – Peak distribution of Profile 1

Profile 2 – Unsaturation profile

Using the accurate mass detected and the elemental composition tool, molecules having 27 carbons with different unsaturation degrees are reported. Figure 7 shows the peak area profile of this group. Table 3 shows the empirical formula and calculated exact mass.

Figure 7 – Peak distribution of Profile 2

Conclusion

Luxon Ion Source combined with a Q-Exactive system allows ultra-fast (8 seconds per sample) analysis of hydrocarbon (C20 to C80) in different petroleum samples.

The post Analysis Method for Hydrocarbons Profile appeared first on Phytronix.

The European Union has decided to ban boar castration by 2018. As hundreds of millions of boars are slaughtered every year for meat consumption, there is a need for methods of detection of boar taint. Compounds responsible for boar taint include androstenone, indole and skatole.

We propose to perform a fast, cheap and full automated sample preparation method followed by a quantification using Laser Diode Thermal Desorption Mass Spectrometry (LDTD-MS/MS), an ultra-fast quantification technique.

Luxon Ionization Source

The Luxon Ion Source® (Figure 1) is the second-generation sample introduction and ionization source based on the LDTD® technology for mass spectrometry. Luxon Ion Source® uses Fiber-Coupled Laser Diode (Figure 2) to obtain unmatchable thermal uniformity giving more precision, accuracy and speed. The process begins with dry samples which are rapidly evaporated using indirect heat. The thermally desorbed neutral molecules are carried into a corona discharge region. High efficiency protonation and strong resistance to ionic suppression characterize this type of ionization and is the result of the absence of solvent and mobile phase. This thermal desorption process yields high intensity molecular ion signal in less than 1 second sample to sample and allows working with very small volumes.

Figure 1 – Luxon Ion Source®

Figure 2 – Schematic of the Luxon Ionization Source

Sample Preparation Method

Automated System

Figure 3 – Automated sample preparation and analysis of Indole, Skatole and Androstenone

Extraction method

• 0.3 g of back fat sample in 8 positions custom dounce receptacle. Transfer to homogenization section.
• Dounce homogenization and solvent addition:
  • 4 mL NaOH (1N) containing NaCl.
  • 3 mL internal standard mixture (200 ng/mL Androstenone-d₄ and 5.4 ng/mL Skatole-d₃ in MTBE).
• Mixing by Up and Down.
• Wait 1 minute for phase separation.
• Transfer 5 µL to Lazwell plate.
• Wait 1 minute for drying completely.
• Plate transfer to Luxon Ion Source and analyze by LDTD-MS/MS.
• Automatic data processing to give a GO or NO-GO sample analysis based on cut-off levels.

LDTD-MS/MS Parameters

LDTD

Model: Phytronix, LDTD® S-960, with LazWell Infeed System

Carrier gas: 6 L/min (air)

Laser pattern:

• 1 second start delay
• 3 seconds ramp to 65% power

MS/MS

Model: Sciex 5500 Qtrap system®

Ionization: APCI

Ionization mode: Positive

Curtain: 20

Time: 4 seconds

Results and Discussion

Linearity

Negative back fat sample extracts are spiked to get the following calibration range around the proposed sorting thresholds: 332.5 to 2660 ng/g for Androstenone, 41.3 ng/g to 660 ng/g for Skatole and 16.5 ng/g to 132 ng/g for Indole. Correlation coefficients are equal or greater than 0.99 for the quantification curve of each molecule. The LLOQ is greater than the required concentration of boar taint analysis.

A

B

C

Figure 4 – Standard curve for Androstenone (A), Skatole (B) and Indole (C)

Precision

Spiked samples around the decision point and blank solutions are used to validate the precision of the method. Each concentration must not exceed 20 %CV and the mean concentration ± 2 times the standard deviation (± 2 SD) must not overlap with other concentrations at the decision point. The peak area against IS ratio was used to normalize the signal. Replicate extractions are deposited on a LazWell plate and dried before analysis. No overlapping at the decision point is observed for all curves and the CV% was below 15%. Results using the ± 2 SD overlay are plotted. Figure 5 shows the results.

A

B

C

Figure 5 – Precision curve for Androstenone (A), Skatole (B) and Indole (C)

Dry Stability of Samples Spotted in LazWell

Androstenone, Skatole and Indole are volatile compounds and their dry stability in a LazWell kept at room temperature is evaluated. Extracted samples are spotted onto a LazWell plate and kept 1 hour at room temperature before analysis. The reproducibility and accuracy are reported in Table 2 for LLOQ samples. All the results are within the acceptable range (criteria %CV ≤20% and %Nominal 100 ± 20%) with less than 20% blank interference for 1 hour at room temperature.

Conclusion

Luxon Ion Source® based on the LDTD technology combined with a Sciex 5500 Qtrap system allows ultra-fast (8 seconds per sample) and accurate quantification of Androstenone, Skatole and Indole in back fat sample using a full automated sample preparation process.

The post Automated Process for Analysis of Indole, Skatole and Androstenone in Pork appeared first on Phytronix.

Since the hair root is vascularized during its growth, illicit drugs present in the blood stream may enter the hair shaft via the root where they will be sequestered. Therefore, the use of illicit drugs can be revealed by analyzing a small hair sample. To increase the analysis throughput of hair samples, the Luxon Ion Source® coupled to tandem mass spectrometry (MS/MS) was used for the identification and quantification of drugs of abuse.

For this project, we propose to perform a generic extraction method for illicit drug analysis in hair. Screening using the Luxon coupled to a mass spectrometer (Luxon-MS/MS) is chosen as a fast-analytical technique.

Luxon Ionization Source

The Luxon Ion Source® (Figure 1) is the second-generation sample introduction and ionization source based on the LDTD® technology for mass spectrometry. Luxon Ion Source® uses Fiber-Coupled Laser Diode (Figure 2) to obtain unmatchable thermal uniformity giving more precision, accuracy and speed. The process begins with dry samples which are rapidly evaporated using indirect heat. The thermally desorbed neutral molecules are carried into a corona discharge region. High efficiency protonation and strong resistance to ionic suppression characterize this type of ionization and is the result of the absence of solvent and mobile phase. This thermal desorption process yields high intensity molecular ion signal in less than 1 second sample to sample and allows working with very small volumes.

Figure 1 – Luxon Ion Source®

Figure 2 – Schematic of the Luxon Ionization Source

Sample Preparation Method

A pre-wash of the hair is performed to remove external contaminants using Methanol. 10 mg of hair cut into small pieces are transferred in a vial.

2 mL of methanol containing TFA at 0.5% (with internal standard) is added and samples are soaked at 60 degrees Celsius for 1h45. Samples are then sonicated for 15 minutes.

After the extraction, 500 µL of sample are mixed with 200 µL of a solution of KH₂PO₄ (1 mM) / BSA (100 µg/mL) in water.

8 μL of the extract are spotted into 96-LazWell plates and evaporated to dryness at 40 degrees Celsius for 8 minutes. Luxon-MS/MS analysis is done after a complete evaporation.

LDTD-MS/MS Parameters

LDTD

Model: Phytronix, Luxon S-960

Carrier gas: 6 L/min (air)

Laser pattern: 3 second ramp to 55% power and hold 2 seconds

MS/MS

Model: Q-Trap System® 5500, Sciex

Ionization: APCI

Results and Discussion

Precision

Spiked samples around the decision point and blank solutions are used to validate the precision of the method. Each concentration must not exceed 20% CV and the mean concentration ± 2 times the standard deviation must not overlap with other concentrations at the decision point. The peak area against IS ratio was used to normalize the signal. Replicate extractions are deposited on a LazWell plate and dried before analysis. No overlapping at the decision point is observed for all curves and the CV% was below 15% for within-run experiments. Results using the ± 2 STD overlay are plotted. Figure 3 shows the results of the within-run test for amphetamine. Similar results are obtained for the other drugs.

Figure 3 – Within run Precision curves for Amphetamine

For the inter-run precision experiment, each fortified sample sets are analyzed in triplicate on five different days. Table 3 shows the inter-run precision results.

Wet stability of sample extracts

Following the extraction, sample extracts are kept at 4°C in closed containers. After 4 days, sample extracts were spotted on a LazWell plate and analyzed. Precision at 50% cut-off standard is reported in Table 4 for Amphetamine. All the results are within the acceptable range (criteria %CV ≤20%) for 4 days at 4°C. Similar results are obtained for the other drugs.

Dry Stability of Samples Spotted in LazWell

Extracted samples are spotted onto a LazWell plate and kept at room temperature before analysis. Precision at 50% cut-off standard is reported in Table 4 for Amphetamine. All the results are within the acceptable range (criteria %CV ≤20%) for 2 hours at room temperature. Similar results are obtained for the other drugs.

Luxon-MS/MS: Sample screen

Sample specimens are extracted and analyzed using a Luxon-MS/MS method. After a fast desorption, specimens, fortified and blank samples are evaluated using peak area ratio. All samples having a concentration higher than the cut-off standard are classified as drug positive samples. Table 5 shows the screening and confirmation results of the samples. All samples are analyzed using LC-MS/MS confirmation method for cross validation. No false positives or false negatives are observed using the Luxon-MS/MS screening method.

Conclusion

Luxon Ion Source® combined to Q-Trap 5500 mass spectrometer system allows ultra-fast (8 seconds per sample) screening of drugs in Hair sample using a generic sample preparation.

Table 5 – Sample screen and confirmation results

Note: High concentrations of Hydrocodone (Codeine isobar drug) are detected in that sample by the confirmation method.

The post Screening of in hair for drugs with Sciex MS appeared first on Phytronix.

Neuroleptics, also known as antipsychotic medication, are used to treat and manage symptoms of many psychiatric disorders. They can be divided into two classes: first-generation or “typical” antipsychotics and second-generation or “atypical” antipsychotics. Both generations of medication tend to block receptors in the brain’s dopamine pathways, but atypical tend to act on serotonin receptors as well. To increase the analysis throughput of plasma samples, the Luxon Ion Source® coupled to tandem mass spectrometry (MS/MS) was used for the quantification of neuroleptic drugs.

For this project, we propose to perform a generic extraction method for neuroleptic drug analysis in plasma. Quantification using the Luxon coupled to a mass spectrometer (Luxon-MS/MS) is chosen as a fast-analytical technique.

Luxon Ionization Source

The Luxon Ion Source® (Figure 1) is the second-generation sample introduction and ionization source based on the LDTD® technology for mass spectrometry. Luxon Ion Source® uses Fiber-Coupled Laser Diode (Figure 2) to obtain unmatchable thermal uniformity giving more precision, accuracy and speed. The process begins with dry samples which are rapidly evaporated using indirect heat. The thermally desorbed neutral molecules are carried into a corona discharge region. High-efficiency protonation and strong resistance to ionic suppression characterize this type of ionization and is the result of the absence of solvent and mobile phase. This thermal desorption process yields high-intensity molecular ion signal in less than 1 second sample-to-sample and allows working with very small volumes.

Figure 1 – Luxon Ion Source®

Figure 2 – Schematic of the Luxon Ionization Source

Sample Preparation Method

Neuroleptics multilevel plasma calibrator set from Chromsystems containing the following drugs is used: Aripiprazole, Dehydroaripiprazole, Clozapine, Desmethylclozapine, Haloperidol, Olanzapine N-desmethylolanzapine, Quetiapine, Norquetiapine, Risperidone and 9-Hydroxyrisperidone. These are used as calibration standards. Control Level I and Level II from Chromsystems are used as quality control samples.

Protein precipitation is performed by mixing 20 µL plasma, 4 µL internal standard solution and 200 µL acetonitrile. Vortex and centrifuge at 14000 rpm for 1 minute. Finally, 5 µL of the upper layer are spotted into 96-LazWell-DEC plates and evaporated to dryness. Luxon-MS/MS analysis is performed after a complete evaporation.

LDTD-MS/MS Parameters

LDTD

Model: Phytronix, Luxon S-960

Carrier gas: 6 L/min (air)

Laser pattern: 6 second ramp to 65% power and hold 2 seconds.

MS/MS

Model: Q-Trap System® 5500, Sciex

Ionization: APCI (Positive)

Results and Discussion

Linearity

Calibrator set spiked at the following calibration ranges are extracted: 20.6 to 746 µg/L for Aripiprazole, 7.56 to 273 µg/L for Dehydroaripiprazole, 77.7 to 1149 µg/L Clozapine, 75.1 to 453 µg/L for Desmethylclozapine, 2.03 to 30.03 µg/L for Haloperidol, 3.85 to 153 µg/L for Olanzapine, 3.92 to 160 µg/L for N-Desmethylolanzapine, 27.3 to 525 µg/L for Quetiapine, 11 to221 µg/L for Norquetiapine, 2.04 to 30.7 µg/L for Risperidone and 3.68 to 138 µg/L for 9-Hydroxyrisperidone. Correlation coefficients are equal or greater than 0.99 (Table 2) for the quantification curve of each molecule. Figure 3 shows the calibration curve of Clonidine. Similar results are obtained for the other Neuroleptic drugs. Figure 4 shows the typical desorption peak of Clonidine with a 0.16-minute window.

Figure 3 – Clozapine calibration curve

Figure 4 – Clozapine desorption peak

Precision and accuracy

Replicate extractions of control Level I and II are deposited on a LazWell plate and dried before analysis. Six replicas are analyzed for within-run evaluation. CV% was below 15% and accuracy within ±20% of the nominal concentration is reached. Table 3 shows the intra-run precision/accuracy results.

For the inter-run precision/accuracy experiment, each control sample sets are analyzed in sextuplicate on three different days. Table 3 shows the inter-run precision/accuracy results.

Conclusion

Luxon Ion Source® combined to Q-Trap 5500 mass spectrometer system allows ultra-fast (10 seconds per sample) quantification of neuroleptic drugs in plasma using a generic protein precipitation extraction procedure.

The post Neuroleptics drugs in Plasmas appeared first on Phytronix.

Legalization of cannabis in various US states and other countries lead to a development of efficient analytical tool for law-enforcement officers to evaluate D⁹-tetrahydrocannabinol (THC) (major active metabolite of Cannabis). When consumption ends, THC levels in blood decrease rapidly, giving a short period of time for police enforcement to collect blood.

For this project, we propose to perform a liquid-liquid extraction method for THC in blood collected with a Mitra device. Quantification using the Luxon coupled to a mass spectrometer (Luxon-MS/MS) is chosen as a fast-analytical technique.

Luxon Ionization Source

The Luxon Ion Source® (Figure 1) is the second-generation sample introduction and ionization source based on the LDTD® technology for mass spectrometry. Luxon Ion Source® uses Fiber-Coupled Laser Diode (Figure 2) to obtain unmatchable thermal uniformity giving more precision, accuracy and speed. The process begins with dry samples which are rapidly evaporated using indirect heat. The thermally desorbed neutral molecules are carried into a corona discharge region. High efficiency protonation and strong resistance to ionic suppression characterize this type of ionization and is the result of the absence of solvent and mobile phase. This thermal desorption process yields high intensity molecular ion signal in less than 1 second sample-to-sample and allows working with very small volumes.

Figure 1 – Luxon Ion Source®

Figure 2 – Schematic of the Luxon Ionization Source

Sample Preparation Method

THC is spiked in human EDTA-k2 blood at concentrations ranging from 2.5 to 100 ng/mL. 10 µL blood samples are then collected with a Mitra micro sampling device (Figure 3).

The blood collector part of the device is transferred in a glass tube (10X75mm) containing 100 µL EDTA buffer (500 µg/mL). Tubes are put in a sonicator bath for 10 minutes. 10 µL of internal standard (THC-d3, 500 ng/mL in acetonitrile) and 100 µL Hexane:Ethyl Acetate (90:10) are added. The solution is then vortexed and centrifuged at 5000 rpm for 2 minutes. Finally, 8 µL of the upper layer are spotted into LazWell96 plates and evaporated to dryness. Luxon-MS/MS analysis is performed after a complete evaporation.

Figure 3 – Mitra micro sampling device (10 µL)

LDTD-MS/MS Parameters

LDTD

Model: Phytronix, Luxon T-960

Figure 4 – LUXON parameters

MS/MS

Model: TSQ Altis plus, Thermo Fisher Scientific

Ionization: APCI (negative)

Results and Discussion

Linearity

A THC calibrator set spiked at the following calibration range are extracted: 2.5 to 100 ng/mL. Correlation coefficients are equal or greater than 0.99 for the quantification curve of THC. Figure 5 shows the calibration curve of THC. Figure 6 shows a typical THC desorption peak with a 0.16-minute window.

Figure 5 – THC calibration curve

Figure 6 – THC desorption peak

Precision and accuracy

Replicate extractions of control (LLOQ, QC-Low, QC-Medium, QC-High and ULQC) are deposited on a LazWell plate and dried before analysis. Replicas are analyzed for inter-run evaluation. CV% below 15% and accuracy within ±15% of nominal concentration are reached. Table 2 shows the inter-run precision/accuracy results.

THC recovery on Mitra device

Spiked samples of THC (100 ng/mL) are added on a Mitra device (10 µL). The concentration of THC is evaluated against a calibration curve where 10 µL of spiked blood sample are added directly in an extraction tube (100% Mitra recovery). A recovery of 71.5% is obtained. Results are shown in Table 3.

THC stability

THC is a hydrophobic and unstable drug. Different stability parameters are verified. All stability results are shown in Figure 7.

Wet stability in buffer at room temperature and 4°C

After blood collection with the Mitra device, absorbent parts are transferred in glass tubes containing an EDTA buffer. The tubes are kept at room temperature for 1 and 3 hours, then extracted. After 3 hours, in the buffer at room temperature, the THC concentration drops below 80% of the nominal value, but the results after 1 hour are within acceptable nominal values (between 85% to 115%). The same experiments are performed at 4°C for 1, 3 and 20 hours. After 20 hours, in the buffer at 4°C, the THC concentration is still within the acceptable nominal value range.

Wet stability in buffer and extraction solvent at 4°C

After a complete extraction with the Mitra device, the mixture of absorbent parts, EDTA buffer and extraction solvent is kept at 4°C for 1, 3 and 20 hours. After 20 hours, the THC concentration is still within the acceptable nominal value range (between 85% to 115%).

Dry stability of samples on Mitra device

Blood samples are collected with the Mitra device and kept at room temperature for 1, 3 and 20 hours. Then, the absorbent parts are transferred in glass tubes containing the EDTA buffer and an LLE is performed. After 3 hours (blood sample on Mitra device at room temperature), the THC concentration drops below 80% of the nominal value, but the results after 1 hour are within the acceptable nominal value (between 85% to 115%) value.

Figure 7 – THC stability results

Conclusion

A low blood sample volume (10 µL) can be properly collected with the Mitra device for THC analysis. THC analysis needs particular care when dealing with the stability of samples. Luxon Ion Source® combined to Thermo TSQ Altis plus mass spectrometer system allows ultra-fast (8 seconds per sample) quantification of THC.

The post Blood Collection of THC Samples and Analysis appeared first on Phytronix.

Many countries worldwide are currently experiencing a public health crisis due to the abuse of illicitly manufactured fentanyl (IMF) and its analogues. Unfortunately, IMF and its analogues are not always part of routine toxicology testing. Thus, there is an urgent need for developing sensitive screening tools for urine samples.

Our goal for this application note is to use a Liquid-Liquid Extraction sample preparation method for the screening of all fentanyl analogues in a single operation in LDTD-MS/MS.

LDTD-MS/MS offers specificity combined with an ultra-fast analysis for an unrivaled screening method. To develop this application, we focused on performing a quick and simple preparation method. Fourteen (14) illicitly manufactured fentanyl (IMF) are analyzed simultaneously with quantitative screening results obtained in less than 9 seconds per sample. Specific cut-off values were attained for each individual drug.

Luxon Ionization Source

The Luxon Ion Source® (Figure 1) is the second-generation sample introduction and ionization source based on the LDTD® technology for mass spectrometry. Luxon Ion Source® uses Fiber-Coupled Laser Diode (Figure 2) to obtain unmatchable thermal uniformity giving more precision, accuracy and speed. The process begins with dry samples which are rapidly evaporated using indirect heat. The thermally desorbed neutral molecules are carried into a corona discharge region. High efficiency protonation and strong resistance to ionic suppression characterize this type of ionization and is the result of the absence of solvent and mobile phase. This thermal desorption process yields high intensity molecular ion signal in less than 1 second sample to sample and allows working with very small volumes.

Figure 1 – Luxon Ion Source®

Figure 2 – Schematic of the Luxon Ionization Source

Sample Preparation Method

• Add 50 µL urine sample
• Add 5 µL internal standard (Fentanyl-d₅ and Norfentanyl-d₅, 100 ng/mL and 300 ng/mL in MeOH:Water/1:1).
  • Vortex
• Add 50 µL NaOH (0.1N)
• Add 200 µL MTBE
  • Vortex
• Spot 5 µL upper-layer phase on a LazWell96 plate
  • Dry 1 minute at room temperature
• LDTD-MS/MS analysis

LDTD®-MS/MS Parameters

LDTD

Model: Luxon S-960, Phytronix

Carrier gas: 3 L/min (air)

Laser pattern:

• 3-second ramp to 65% power
• Hold 2 seconds at 65% power

MS/MS

MS model: Q-Trap System® 5500, Sciex

Scan Time: 5 msec

Total run time: 8 seconds per sample

Ionization: APCI

Analysis Method: Positive MRM mode

Results and Discussion

Precision

Spiked samples around the decision point and blank solutions are used to validate the precision of the method. Each concentration must not exceed 20% CV and the mean concentration ± 2 times the standard deviation must not overlap with other concentrations at the decision point. The peak area against the internal standard (IS) ratio was used to normalize the signal. Replicate extractions are deposited on a LazWell plate and dried before analysis. No overlapping at the decision point is observed for all curves and the CV% was below 15% for intra-run experiments. Results using the ± 2 STD overlay are plotted at Figure 3. The results of intra-run test for Fentanyl allow a cut-off value of 1 ng/mL in urine. All cut-offs of different drugs are determined this way.

Figure 3 – Intra-Run Precision Curves for Fentanyl

For the inter-run precision experiment, each fortified sample sets are analyzed in triplicate on five different days. Table 2 shows the inter-run precision results.

Linearity

Blank urine sample is spiked to get different concentration to generate a calibration curve. Table 3 shows the inter-day correlation coefficients for all drugs. Values greater than 0.99 are obtained for all drugs. Figure 4 shows typical calibration curve results for Fentanyl.

Figure 4 – Standard Curve for Fentanyl

Wet Stability of Sample Extracts

Following the extraction, sample extracts are kept at 4°C in closed containers. After 4 days, sample extracts were spotted on a LazWell plate and analyzed. Precision at 50% cut-off standard is reported in Table 4 for Fentanyl. All the results are within the acceptable range (criteria %CV ≤20%) for 4 days at 4°C. Similar results are obtained for the other drugs.

Dry Stability of Samples Spotted in LazWell

Extracted samples are spotted onto a LazWell plate and kept at room temperature before analysis. Precision at 50% cut-off standard is reported in Table 4 for Fentanyl. All the results are within the acceptable range (criteria %CV ≤20%) for 2 hours at room temperature. Similar results are obtained for the other drugs.

Conclusion

Luxon Ion Source® combined to Sciex Q-Trap 5500 mass spectrometer system allows ultra-fast (8 seconds per sample) screening of illicitly manufactured fentanyl (IMF) and its analogue drugs in urine sample using a simple generic sample preparation method.

The post Fentanyl Analogs in Urine at 8 seconds per sample appeared first on Phytronix.