Vitamin K1 (phylloquinone) is an essential compound involved in the synthesis of several blood coagulation factors, bone metabolism, and the prevention of vascular calcification. Therapeutic monitoring, particularly in patients on anticoagulants, is crucial for optimizing treatment and ensuring effective care.

Our goal for this application is to use a combination of efficient sample preparation for the quantification of vitamin K1 in serum using the Luxon Ion Source®, based on LDTD technology.

LDTD-MS/MS offers specificity combined with an ultra-fast analysis for an unrivaled quantification method. To develop this application, we focused on performing efficient sample preparation. Vitamin K1 is analyzed, and results are obtained in less than 10 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 saturation 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

Due to the instability of vitamin K1, stock solutions were prepared in ethanol (0.01% BHT). Pooled serum was exposed to UV light for at least 24h due to photodegradation of endogenous vitamin K. The pooled exposed serum was then spiked to generate a calibration curve.

• 5 µL of working standard solution (10X) is added to a micro-centrifugation tube of 0.5 mL.
• 5 µL of Internal standard (Phylloquinone-D₇, 100 ng/mL in EtOH (0.01% BHT) were added to each sample.
  • Mix 10 seconds at 1100 RPM.
• 50 µL of serum sample or negative matrix for calibration curve.
  • Mix 10 seconds at 1100 RPM.
• 400 µL of isopropanol (IPA) were added.
  • Mix 30 seconds at 1100 RPM.
• Transfer 200 µL of upper layer into a 10 X 75 mm borosilicate tube.
  • Evaporate to dryness (0.5 PSI at 40°C for 15 minutes).
• Reconstitute with 200 µL of Hexane.
  • Mix 10 seconds at 1100 RPM.
• Transfer 100 µL of upper layer into a 6 X 31 mm borosilicate tube.
  • Evaporate to dryness (10 LPM at40°C for 5 minutes).
• Reconstitute with 100 µL of a mixture of isopropanol/water (80/20).
  • Mix 10 seconds at 1100 RPM.
• Spot 5 µL of mixture onto a LazWell 96 plate.
  • Dry 3 minutes at 40°C.

LDTD®-MS/MS Parameters

LDTD

Model: Luxon SH-960, Phytronix

Carrier gas: 9 L/min

Laser pattern:

• 9-second ramp to 45% power*

*Note: Power must be optimized with each instrument

MS/MS

MS model: Triple Quadrupole LCMS-8060, Shimadzu

DL Temperature: 250°C

Heat Block Temperature: 250°C

Voltage: 3500 V

Dwell Time: 10 msec

Total run time: 10 seconds per sample

Analysis Method: Negative MRM mode

Table 1 – MRM transitions for LDTD-MS/MS

Results and Discussion

Validation Test

Calibration curves ranging from 250 to 5000 pg/mL were prepared in a photodegraded human serum. Replicate extractions were deposited onto a LazWell plate and dried before analysis. The peak area against the internal standard (IS) ratio was used to normalize the signal.

Linearity

The calibration curves were plotted using the peak area ratio and the nominal concentration of standards. For the linearity test, the following acceptance criteria was used:

• Linear regression (determination coefficient, r²) must be ≥ 99

Table 2 shows the inter-day correlation coefficients for Vitamin K1. Determination coefficient (r²) values greater than 0.99 are obtained. Figure 3 shows a typical calibration curve result for Vitamin K.

Figure 3 – Vitamin K1 calibration curve

Table 2 – Inter-day calibration curve correlation coefficients (r²)

Precision and Accuracy

For the accuracy and precision evaluation, the following acceptance criteria were used:

• Each concentration must not exceed 15% CV
• Each concentration must be within 100 ± 15% of the nominal concentration

For the intra and inter-run precision and accuracy experiment, each QC was analyzed in sextuplicate, on five different days. Table 3 and 4 shows the intra and inter-run precision and accuracy results for Vitamin K1. The obtained %CV was below 15% and the accuracy was within 15% of the nominal value.

Table 3 – Intra-Run Precision and Accuracy of Vitamin K1

Table 4 – Inter-Run Precision and Accuracy of Vitamin K1

Wet Stability of Sample Extracts

Following the extraction, sample extracts are kept at 4°C in closed containers protected from light. After 24 hours, sample extracts are spotted on a LazWell plate, dried and analyzed. Precision and accuracy of QC samples are reported in  Figure 4. All the results are within the acceptable criteria range for 24 hours at 4°C.

Dry Stability of Samples Spotted in LazWell

Extracted samples are spotted onto a LazWell plate, dried and kept at room temperature for 1 hour before analysis. The precision and accuracy results of QC samples are reported in Figure 4. All the results are within the acceptable criteria range for 1 hour at room temperature.

Figure 4 – Wet and Dry Stability of Vitamin K1

Cross validation study

Real patients’ serum samples (N=8) have been tested with this method to correlate with results obtained by traditional LC-MS/MS. The percentage of difference between the values are evaluated. A difference below 20% is obtained. Results are reported in Table 5.

Table 5 – Comparison between Vitamin K1 concentration values

Conclusion

The Luxon Ion Source® combined with Shimadzu Triple Quadrupole LCMS-8060 mass spectrometer system allows the ultra-fast (10 seconds per sample) analysis of Vitamin K1 in serum.

The post Analysis of Vitamin K1 in Serum appeared first on Phytronix.

Oral fluid drug testing is an increasingly popular method for detecting substance abuse, due to the ease and speed of sample collection. However, if the sample is not taken directly by an official, it is not possible to know the integrity of the sampling and/or the sample.

The objective of this application is to develop a rapid quantification method to analyze cholesterol in oral fluid using LDTD-MS/MS to assess sample integrity and detect samples that have never been in contact with oral fluid.

The LDTD-MS/MS system offers specificity combined with ultra-rapid analysis for the analysis of cholesterol in oral fluid samples. To develop this application, we focused on making sample preparation simple and fast. LDTD-MS/MS analysis is performed in less than 8 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 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

Sample Collection

Oral fluids were collected using the Quantisal® device. After the collection of the oral fluid, the pad is transferred into a tube containing an extraction buffer. During this process, oral fluids are diluted by a factor of 4. To quantify cholesterol, oral fluid is replaced by water in the calibration curve.

Figure 3 – Quantisal device for oral fluid sample collection (1)

Sample Extraction

• In a borosilicate tube (12X75 mm), 10 µL of Internal standard in methanol is added
• 100 µL of OF sample mixed with Quantisal buffer (1/3) are added
• 25 µL of methanol are added (for calibration curve and QCs, working solution 1X in methanol are added)
  • Vortex 30 seconds
• 500 µL of extraction solution are added (DCM/Cl₃CH/Isopropanol/Heptane (80/25/8.5/16.5)
  • Vortex (1000 rpm/90 seconds)
  • Centrifuge (5000 rpm/5 min)
• In a second borosilicate tube (12X75 mm) mix 300 µL of the bottom layer with 75 µL of the evaporation solution (1% acetic acid in methanol).
  • Evaporate until dryness with gentle airflow at room temperature (RT)
• Add 40 µL of acetonitrile to reconstitute samples.
  • Vortex 30 seconds
• Add 40 µL of desorption solution
  • Vortex 30 seconds
• Spot 4 µL of mixture on a LazWell96 plate
  • Dry 8 minutes at RT

LDTD®-MS/MS Parameters

LDTD

Model: Luxon S-960, Phytronix

Carrier gas: 6 L/min (air)

Laser pattern:

• 1-second start delay
• 6-second ramp to 65% power

MS/MS

MS model: QTRAP 5500, Sciex

Scan Time: 50 msec

Ionization: APCI

Analysis Method: Positive ionization mode

Table 1 – MRM transitions for LDTD-MS/MS

Results and Discussion

Initial Cut-off Test (ng/mL)

Linearity

Samples were spiked at 0.5 µg/mL to 5 µg/mL. Standard working solutions were prepared in methanol. Calibration points were extracted in triplicate to obtain a coefficient of variation higher than 0.99.

Table 2 – Coefficient of correlation obtain for 6 runs.

Precision and Accuracy

Each calibration point was used to validate the precision and the accuracy 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 standard must not exceed 15%CV for at least 66.6% of samples.
• Each standard must not exceed 15% Bias for at least 66.6% of samples. 

For intra-run precision and accuracy experiment, a fortified sample set in water was extracted and analyzed in triplicate. Table 3 shows intra-run results, %CV and % Bias obtained were lower that 15 % for each concentration analyzed. For the inter-run precision experiment, each fortified sample set is analyzed in triplicate on four different days. Table 4 shows the inter-run precision and accuracy results. %CV and %Bias were below 15%.

Table 3 – Intra-Run Precision and Accuracy Results

Table 4 – Inter-Run Precision and Accuracy Results

Wet Stability of Sample Extracts

Following the extraction, the extracted sample was mixed with a desorption solution and kept at 4°C in closed containers. After 1 day, extracted samples are spotted on a LazWell plate, dried and analyzed. Sample precision and accuracy are reported in Figure 4. All the results are within the acceptable criteria range for 2 days at 4°C.

Dry Stability of Samples in LazWell

Extracted samples are spotted onto a LazWell plate, dried and kept at room temperature for 1 hour and 2 hours before analysis. The precision and accuracy results are reported in Figure 4. All the results are within the acceptable criteria range for 2 hours at room temperature.

Figure 4 – Wet and Dry stability evaluation: A) Precision results B) Accuracy results

Multi-Matrix validation

Five different oral fluid matrices were analyzed by LDTD-MS/MS and by LC-MS/MS. Samples were collected using Quantisal device than extracted using the method previously mentioned. Multi-matrix validation results are shown in Table 5. The difference (in %) between the two instrumental methods were lower than 20 % for all five matrices.

Table 5 – Multi-matrix validation for cholesterol (n=1)

Integrity evaluation

To identify false oral fluid samples that can be submitted by patients, two false samples were introduced into the matrices lot and all samples were extracted. One sample was dipped in water before closing the Quantisal collection device (absorbed water instead of oral fluid) and one sample did not absorb any water or oral fluid and was placed directly into the Quantisal collection device. The cholesterol concentration must be higher than 0.5 µg/mL to be considered a real sample. Results are presented in Table 6.

Table 6 – Integrity evaluation of Oral fluid sample

Conclusion

Luxon Ion Source® combined to a Sciex QTRAP 5500 mass spectrometer system allows ultra-fast (8 seconds per sample) for evaluating the integrity of Oral fluid samples using a simple sample preparation method.

Reference

1. Quest Diagnostics, Drug monitoring oral fluid collection [Online], URL: https://www.questdiagnostics.com/healthcare-professionals/about-our-tests/drug-testing/oral-fluid-collection (accessed on 2024-05-23).

The post Assessing the Integrity of an Oral Fluid Sample appeared first on Phytronix.

Glipizide, a second-generation of sulfonylurea, is effective in controlling the blood glucose in patients with noninsulin-dependent diabetes mellitus¹. During antidiabetic therapy, it is critical to monitor the plasma concentration of glipizide.

Our goal for this application note is to use an automated sample preparation method for the quantification of Glipizide in plasma using a single operation in LDTD-MS/MS.

LDTD-MS/MS offers specificity combined with an ultra-fast analysis for an unrivaled quantification method. To develop this application, we focused on performing a quick and simple sample preparation. Glipizide is analyzed, and results are obtained in less than 5 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 saturation 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

Glipizide, stock solutions were prepared in methanol. EDTA-K2 plasma was used as a negative matrix then spiked to generate a calibration curve and QCs. 

Plasma samples were transferred into barcoded tubes, readable by the Azeo extraction system. Each barcoded vial was scanned by the Azeo Liquid Handler and an automatic batch file was created.

The Azeo Liquid Handler (Figure 3) is used to extract the samples using the following conditions:

• 10 µL of Internal standard (Glipizide-d₁₁ at 1000 ng/mL in Acetonitrile:Water (1:1)) were added to a deep-well plate placed on the Lumo Vortexer.
• 50 µL of plasma sample were transferred from the vials to a deep-well plate placed on the Lumo Vortexer.
  • Mix (30 seconds at 1000 rpm).
• 50 µL of extraction buffer were added into a deep-well plate.
  • Mix (30 seconds at 1000 rpm).
• 300 µL of Methyl-Tert-Butyl Ether (MTBE) were added into a deep-well plate.
  • Mix (30 seconds at 1000 rpm).
  • Centrifugation at 5000 rpm for 2 minutes for phase separation.
• Spot 7 µL upper layer phase onto a coated LazWell 96 plate.
  • Dry at room temperature.

Figure 3 – Automated extraction system.

LDTD®-MS/MS Parameters

LDTD

Model: Luxon SH-960, Phytronix

Carrier gas: 3 L/min (air)

Laser pattern:

• 3-second ramp to 65% power

MS/MS

MS model: LCMS-8060, Shimadzu

Scan Time: 50 msec.

Total run time: 5 seconds per sample

Ionization: APCI

Analysis Method: Positive MRM mode

Table 1 – MRM transitions for LDTD-MS/MS

Results and Discussion

Data preparation process

Mass spectrometers are data acquisition systems that were not designed to deal with signals of a few seconds per sample. The synchronization sequence adds 6 to more than 15 seconds between each sample. To bypass this delay, all samples are acquired in a single file (Figure 4). To allow the analysis of such data, Cascade software is designed to detect, split, and integrate every sample peak acquired in a single file.

Figure 4 – Single file mass spectrometer data for 96 samples. Glipizide-d11 transition.

Validation Test

Calibration curves ranging from 10 to 1000 ng/mL and QCs were prepared in EDTA-K2 plasma. Replicate extractions were deposited onto a LazWell plate and dried before analysis. The peak area against the internal standard (IS) ratio was used to normalize the signal.

Linearity

The calibration curves were plotted using the peak area ratio and the nominal concentration of standards. For the linearity test, the following acceptance criteria was used:

• Linear regression (r²) must be ≥ 99

Figure 5 shows a typical calibration curve result for Glipizide. Table 2 shows the coefficients of determination (R²) of six different runs.

Figure 5 – Glipizide calibration curve.

Table 2 – Linearity results

Precision and Accuracy

For the accuracy and precision evaluation, the following acceptance criteria were used:

• Each concentration must not exceed 20% CV.
• Each concentration must be within 100 ± 20% of the nominal concentration.

 For the inter-run precision and accuracy experiment, each QCs were analyzed in triplicate in six different runs. For the intra-run precision and accuracy experiment LLOQ, QC-L, QC-M, QC-H and ULQC were analyzed in six replicates. Table 3 and 4 show the intra and inter-run precision and accuracy results for Glipizide. The obtained %CV was below 15% and the accuracy was within 15% of the nominal value.

Table 3 – Intra-Run Precision and Accuracy

Table 4 – Inter-Run Precision and Accuracy

Stability

Wet stability: Following the extraction, the extracted sample was kept at 4°C in closed containers. After 1 day, extracted samples are spotted on a LazWell plate, dried and analyzed. Precision and accuracy are reported in Table 5. All the results are within the acceptable criteria range for 1 day at room temperature.

Dry stability: Extracted samples are spotted onto a LazWell plate, dried and kept at room temperature for 1 hour before analysis. The precision and accuracy results are reported in Table 5. All the results are within the acceptable criteria range for 1 hour at room temperature.

Table 5 – Wet and Dry Stability of Glipizide

Matrix effect study

Six different plasma samples were spiked at QC-M level, extracted, and analyzed. The precision and accuracy results of the different matrix spiked are reported in Table 6. All the results are within the acceptable criteria range.

Table 6 – Matrix effect result of Glipizide

Conclusion

The Luxon Ion Source® combined with Shimadzu LCMS-8060 mass spectrometer system allows ultra-fast (5 seconds per sample) analysis of Glipizide in plasma.

Reference

1. Brogden et Al. (1979). Drugs 18:329-353

The post Analysis of Glipizide in human plasma appeared first on Phytronix.

Vitamin B3 (Nicotinamide) and B5 (Pantothenic acid) have important roles in physiological functions to maintain good health. Scientific publications report their potential implication in treatments for depression¹. Their role as biomarkers is evaluated. For large epidemiologic studies, high-throughput and accurate analytical techniques are needed.

Our goal for this application note is to use an automated sample preparation method for the quantification of Vitamin B3 and B5 in plasma using a single operation in LDTD-MS/MS.

LDTD-MS/MS offers specificity combined with an ultra-fast analysis for an unrivaled quantification method. To develop this application, we focused on performing a quick and simple sample preparation. Vitamin B3 and B5 are analyzed, and results are 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 saturation 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

Vitamin B3 and B5, stock solutions were prepared in methanol. Bovine serum albumin (BSA) at 10 mg/mL was used as a negative matrix then spiked to generate a calibration curve.

Plasma samples were transferred into barcoded tubes, readable by the Azeo extraction system. Each barcoded vial was scanned by the Azeo Liquid Handler and an automatic batch file was created.

The Azeo Liquid Handler (Erreur! Source du renvoi introuvable.) is used to extract the samples using the following conditions:

• 80 µL of Internal standard (Nicotinamide-13C₆ and Pantothenic acid-13C₃15N at 100 ng/mL and 400 ng/mL in methanol:Water (1:1) were added to a deep-well plate placed on the Lumo Vortexer.
• 80 µL of plasma sample were transferred from the vials to a deep-well plate placed on the Lumo Vortexer.
  • Mix (30 seconds at 1000 rpm).
• 240 µL of acetonitrile were added into a deep-well plate.
  • Mix (30 seconds at 1000 rpm).
  • Centrifugation at 5000 rpm for 2 minutes for phase separation.
• 50 µL of upper layer phase were transferred into a second deep-well plate.
• 50 µL of KH₂PO₄ (4 mM)
• 500 µL Hexane:MTBE (1:1) were added into a deep-well plate
  • Mix (30 seconds at 1000 rpm).
  • Centrifugation at 5000 rpm for 2 minutes for phase separation.
• Spot 4 µL lower layer phase onto a LazWell 96 plate.
  • Dry 5 minute at 40°C.

Figure 3 – Automated extraction system.

LDTD®-MS/MS Parameters

LDTD

Model: Luxon SH-960, Phytronix

Carrier gas: 9 L/min (air)

Laser pattern:

• 6-second ramp to 85% power
• Hold 3 seconds.

MS/MS

MS model: LCMS-8060, Shimadzu

Scan Time: 20 msec.

Total run time: 9 seconds per sample

Ionization: APCI

Analysis Method: Positive MRM mode

Table 1 – MRM transitions for LDTD-MS/MS

Results and Discussion

Data preparation process

Mass spectrometers are data acquisition systems that were not designed to deal with signals of a few seconds per sample. The synchronization sequence adds 6 to more than 15 seconds between each sample. To bypass this delay, all samples are acquired in a single file (Figure 1). To allow the analysis of such data, Cascade software is designed to detect, split, and integrate every sample peak acquired in a single file.

Figure 4 – Single file mass spectrometer data for 96 samples. Pantothenic acid-13C3 15N transition.

Validation Test

Calibration curves ranging from 10 to 1000 ng/mL for Vitamin B3 and B5 were prepared in BSA (10 mg/mL). Replicate extractions were deposited onto a LazWell plate and dried before analysis. The peak area against the internal standard (IS) ratio was used to normalize the signal.

Linearity

The calibration curves were plotted using the peak area ratio and the nominal concentration of standards. For the linearity test, the following acceptance criteria was used:

• Linear regression (r²) must be ≥ 99

Figure 5 shows a typical calibration curve result for Vitamin B3 (A) with a coefficient of determination (r²) of 0.99794 and Vitamin B5 (B) with a r² of 0.99825.

Figure 5 – Vitamin B3 (A) and Vitamin B5 (B) calibration curve.

Precision and Accuracy

For the accuracy and precision evaluation, the following acceptance criteria were used:

• Each concentration must not exceed 20% CV.
• Each concentration must be within 100 ± 20% of the nominal concentration.

For the inter-run precision and accuracy experiment, each standard was analyzed in triplicate in six different runs. Table 2 and 3 show the inter-run precision and accuracy results for Vitamin B3 and B5, respectively. The obtained %CV was below 15% and the accuracy was within 15% of the nominal value.

Table 2 – Inter-Run Precision and Accuracy of Vitamin B3

 Table 3 – Inter-Run Precision and Accuracy of Vitamin B5

Stability

Wet stability: Following the extraction, the extracted sample was kept at room temperature in closed containers. After 1 day, extracted samples are spotted on a LazWell plate, dried and analyzed. Precision and accuracy are reported in Table 3. All the results are within the acceptable criteria range for 1 day at room temperature.

Dry stability: Extracted samples are spotted onto a LazWell plate, dried and kept at room temperature for 1 hour before analysis. The precision and accuracy results are reported in Table 3.  All the results are within the acceptable criteria range for 1 hour at room temperature.

Table 4 – Wet and Dry Stability of Vitamin B3 and B5

Cross validation study

Real patients’ serum samples (N=6) have been tested with this method to correlate with results obtained by traditional LC-MS/MS. The percentage of difference between the values are evaluated. A difference below 20% is obtained. Results are reported in Table 5.

Table 5– Cross validation results

Conclusion

The Luxon Ion Source® combined with Shimadzu LCMS-8060 mass spectrometer system allows ultra-fast (9 seconds per sample) analysis of Vitamin B3 and B5 in plasma.

References

1. Ryan et Al. (2020). Brain, Behavior & Immunity – Health 4, 100063

The post Analysis of Vitamin B3 and B5 in plasma as a Biomarker of Clinical Disorders appeared first on Phytronix.

Gabapentin, a drug originally prescribed for the treatment of seizures, is now frequently used off-label for pain management. However, its use can cause side effects such as drowsiness and dizziness, which may impair driving and pose significant safety risks. Considering these issues, the National Safety Council’s Alcohol, Drugs, and Impairment Division (NSC-ADID) has recently upgraded gabapentin’s classification from a Tier II to a Tier I substance, adding it to the standard routine drug testing panel. For this purpose, NSC-ADID established a cutoff concentration of 50 ng/mL in oral fluid.

Our goal for this application note is to use a simple sample preparation method and a fast analysis technique for gabapentin analysis in oral fluid (OF) sample.

The LDTD-MS/MS system using Axino Ion Source offers specificity combined with an ultra-fast analysis for an unrivaled analysis method. To develop this application, we focused on performing a simple sample preparation.

Sample Preparation Method

Sample Collection

Oral fluid sample collection is performed with the Quantisal device, which is FDA 510(k) cleared for the collection of oral fluid for drug analysis.

Automated Sample Extraction (SALLE)

The calibration curve was prepared in negative oral fluid samples.

• In a 12 X 75 mm borosilicate tubes, 10 µL internal standard solution (gabapentin-d10 at 25 µg/mL in acetonitrile) are added.
• 125 µL of spiked negative OF in Quantisal buffer (1:3) are added.
  • Vortex (1100 rpm/30 seconds)
• 500 µL of extraction solution are added.
• 1000 µL of dilution solution are added.
  • Vortex (1100 rpm/60 seconds)
  • Centrifuge (5000 rpm/5 min.)
• Transfer 50 µL of the aqueous phase (upper layer) in a 0.5 mL microcentrifugation tube.
• In the 0.5 mL microcentrifugation tube, add 150 µL of desorption solution.
  • Vortex (1100 rpm/10 secoonds)
• Spot 5 µL of upper layer on a Domino plate
  • Dry 8 minutes at 40°C

LDTD^®-MS/MS Parameters

LDTD

Model: Axino, Phytronix

Carrier gas: High

Laser pattern: 6-second ramp to 65% power

MS/MS

MS model: Q-Trap System^® 5500, Sciex

Ionization: APCI

Analysis Method: Positive MRM mode

CUR: 20

CAD: 8

Results and Discussion

Validation Test

To evaluate method precision and accuracy, a three-point calibration curve was employed (1 x cutoff, 2 x cutoff, and 5 x cutoff). For the screening approach, a single-point calibration at the cutoff level was used, applying a linear regression constrained to pass through the origin. The peak area against the internal standard (IS) ratio was used to normalize the signal.

Linearity

For inter-run results, linearity was evaluated. Correlation coefficient (r) must be higher than 0.97. Linearity obtained was higher than 0.991.

Inter-run Precision and Accuracy

Spiked samples around the decision point (Cal-1X, two times cut-off: Cal-2X, and five times cut-off: Cal-5X) and blank solutions are used to validate the precision of the method. For quality control samples, these samples are spiked in real negative matrices. For QC-0.5X, 50% cut-off are spike in sample, and for QC-2X, 200% cut-off are spiked in sample. Replicate extractions are deposited on a Domino LazWell plate and dried before analysis.

• Each concentration must not exceed 20% CV for 66.6% of samples
• Each concentration must not exceed 20%Bias for 66.6% of samples.

For the inter-run precision experiment, each fortified sample set is analyzed in triplicate. Table 2 shows the inter-run precision and accuracy results for gabapentin. %CV and %Bias were below 20%.

Run Acceptance Criteria for Intra-run

Intra-run validation tests were carried out using a one-point calibration curve and linear through zero regression. Cutoff and Qcs were analyzed in triplicate. Table 3 shows intra-run results.

• Blank samples must be detected as negative.
• At Cal-1X concentration, %CV must not exceed 20% for at least 66.7% of this standard.
• At QC-0.5X, at least 66.7% of samples must be detected as negative.
• At QC-2X, 66.7% of samples must be detected as positive.

Multi-Matrix Study

Real OF samples (N=4) have been tested with this method. These samples were readily known to be exempt from gabapentin. All samples were determined to be negative by Axino-MS/MS analysis. Results are reported in Table 4

Conclusion

Axino Ion Source^® combined to a Sciex Q-Trap 5500 mass spectrometer system allows ultra-fast (10 seconds per sample) for screening gabapentin in oral fluid using a simple and efficient sample preparation method.

The post Gabapentin Analysis in Oral Fluids appeared first on Phytronix.

The post Axino Ion Source – Product Sheet appeared first on Phytronix.

Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are a group of chemicals made by humans. Since the 1950s, PFASs have been used in many consumer products and industrial processes. They have properties that resist heat, grease, and water. There are thousands of types of PFAS. The most common types and final products of degradation are PFOA (perfluorooctanoic acid) and PFOS (perfluorooctanoic sulfonic acid). They were widely detected in different environmental media (Mahiba Shoeib et al. 2005) and human blood (Perez et al. 2013). PFAS in drinking water is one of the most concerning aspects.

The goal of this application note is to develop an automated sample preparation of drinking water samples and a rapid analytical method to analyze PFAS using LDTD-MS/MS.

Sample Preparation Method

Automated Sample Extraction

Two milliliters of drinking water samples are transferred to a borosilicate tube (12X75 mm) then inserted in the Azeo extraction system (Figure 1). The automated extraction process is as follows:

• Add 20 µL of internal standard solution.
  • Vortex (1100 rpm/30 s)
• Add 30 µL of HCl (4N).
  • Vortex (1100 rpm/30 s)
• Add 700 µL of extraction solution (MTBE).
  • Vortex (1100 rpm/30 s)
  • Phase separation by gravity
• In a sample holder of 96 glass tubes (6X31 mm), add 50 µL ammonium formate (20 mM in methanol).
• Transfer 200 µL of the upper layer into the 6X31 mm glass tube.
  • Vortex (1100 rpm/30 s)
  • Evaporate until dryness (40°C, 10 minutes, air flow 10 LPM).
• Add 60 µL of reconstitution solution.
  • Vortex (1100 rpm/30 s)
• Spot 5 µL of reconstituted sample on a LazWell96 plate.
  • Dry 5 minutes at 40°C.

LDTD^®-MS/MS Parameters

LDTD

Model: Luxon T-960 NG, Phytronix

Carrier gas: 9 L/min (Nitrogen) + 7.5 µL/min TFA solution (0.05% in water)

Laser pattern:

• 6-second ramp to 100% power
• 4-second hold at 100% power

MS/MS

MS model: TSQ Altis plus, Thermo Scientific

IonSpray Voltage: -3800 V

Scan Time: 5 msec

Analysis Method: Negative MRM mode

Results and Discussion

Linearity

The calibration curve is prepared in HPLC water. PFAS concentration between 20 to 200 ng/L are used to evaluate the method linearity and 100 to 10 000 ng/L for PFBA. 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. Figure 4 and Figure 5 shows a typical calibration curve for PFOA and PFOS. Similar results were obtained for the other PFAS.

Precision and Accuracy

Spiked sample solutions are used to validate the precision and accuracy of the method. For the accuracy and precision evaluation, the following acceptance criteria were used:

• Each concentration must not exceed <20% CV.
• Each concentration must be within ± 20% Bias.

For the intra and inter-run precision and accuracy experiment, each fortified sample set is analyzed in sextuplicate, in the same runs. Table 1 shows the intra-run precision and accuracy results for PFOA. %CV was below 20% and the accuracy within 20%. Similar results were obtained for the other PFAS.

For the inter-run precision experiment, each fortified sample set is analyzed in triplicate on three different runs. Table 2 shows the inter-run precision and accuracy results for PFOA. %CV was below 20% and the accuracy within 20%. Similar results were obtained for the other PFAS.

Recovery

Blank samples were extracted and then spiked at the middle calibration level after the automated extraction process. The middle standard was compared to the recovery sample to determine the recovery percentage of PFAS. Table 3 shows the recovery results.

Multi-matrix analysis of PFAS in drinking water

Drinking water was collected from different sites. Samples are analyzed to verify the presence of each PFAS. PFAS are spiked at 100 ng/L (500 ng/L for PFBA) and analyzed as unknown to verify the method performance. Results are report in Table 4.

Conclusion

Luxon Ion Source® combined with a Thermo Scientific TSQ Altis Plus mass spectrometer system allows ultra-fast (10 seconds per sample) analysis of a panel of PFAS in drinking water using a simple and automated sample preparation method.

The post Analysis of PFAS in Drinking Water appeared first on Phytronix.

The determination of serum testosterone and dehydroepiandrosterone (DHEA) concentrations facilitates the diagnosis and treatment of various diseases. In addition to testosterone, the primary androgen hormone, many other androgenic hormones play an important role in the diagnosis and treatment of various diseases. Since the introduction of steroid immunoassays, antibody affinity-based methods have been widely used to measure bioactive steroids in biological fluids due to their high sensitivity. However, these techniques are limited by the specificity of antibodies for epitopes, which may lead to cross-reactions with structurally similar steroids, thereby reducing the specificity of the method. To obtain a rapid analytical method to separate testosterone and DHEA, isobaric compound, a specific APCI ionisation and extraction method and an LDTD-MS/MS analytical method was developed.

Presented during ASMS 2025.

The post High-Throughput Analysis of Testosterone and DHEA in Human Serum appeared first on Phytronix.

Serotonin is an indoleamine neurotransmitter derived from the amino acid tryptophan. Its biological functions are complex, influencing various physiological processes such as mood regulation, memory, and even contributing to undesirable effects like vomiting. The measurement of serotonin concentration in serum is commonly used as a biomarker for diagnosing conditions such as carcinoid syndrome and other related disorders. During method development, the major ion observed is M-2 when using the Laser Diode Thermal Desorption (LDTD) ionization source.

To better understand the ionization process, several experiments are conducted. Ultimately, a reliable method was developed for analyzing serotonin levels in human serum.

Presented at ASMS 2025.

The post Superoxide Radical Anion Oxidative Ionization in Negative LDTD-MS/MS Analysis of Serotonin appeared first on Phytronix.

Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are a group of chemicals made by humans. Since the 1950s, PFASs have been used in many consumer products and industrial processes. They have properties that resist heat, grease, and water. There are thousands of types of PFAS. The most common types and final products of degradation are PFOA (perfluorooctanoic acid) and PFOS (perfluorooctanoic sulfonic acid). They were widely detected in different environmental media (Mahiba Shoeib et al. 2005) and human blood (Perez et al. 2013). The goal of this presentation is to develop an automated sample preparation of drinking water sample and a rapid analytical method to analyze PFAS using LDTD-MS/MS.

Presented at ASMS 2025.

The post From Industry to Tap: Quantifying PFAS in Drinking Water appeared first on Phytronix.