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Diss Factsheets

Toxicological information

Additional toxicological data

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Administrative data

Endpoint:
additional toxicological information
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
January 12, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: No specific test guideline was reported; however, a scientifically defensible investigation approach was used to conduct the study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
no guideline required
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
434-630-6
EC Name:
-
Cas Number:
60372-77-2
Molecular formula:
Hill formula: C20H41N4O3Cl
IUPAC Name:
ethyl N2-dodecanoyl-l-argininate hydrochloride
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material: Nα-Lauroyl-L-arginine ethyl ester monohydrochloride
- Substance type: White powder

Results and discussion

Applicant's summary and conclusion

Conclusions:
Validation of an Liquid Chromatography-MS/MS (13C-LAE,13C-LAS and 13C-arginine) method for the measurement of LAE and two metabolites in human plasma. Full validation consisted of analysis of 2 calibration curves, QC samples (prepared at 1, 3, 15 and 180 ng/ml for C-LAE and C-LAS, prepared at 20, 60, 250 and 900 ng/ml for C-arginine) and analysed on 3 separate occasions to assess precision and accuracy, specificity checks in 6 sources of blank plasma, matrix effects, recovery of analytes and internal standard through extraction, dilution of over-range samples and stability of analytes in plasma and standard solution. In conclusion, an LC-MS/MS method for the measurement of 13C-LAE, 13C-LAS and 13C-arginine in human plasma has been successfully validated for use.
Executive summary:

With the purpose of validating Liquid Chromatography-MS/MS method for the measurement of LAE and two metabolites in human plasma the current study was performed.

Validation consisted of analysis of 2 calibration curves, QC samples (prepared at 1, 3, 15 and 180 ng/ml for 13C-LAE and 13C-LAS, prepared at 20, 60, 250 and 900 ng/ml for 13C-arginine) and analysed on 3 separate occasions to assess precision and accuracy, specificity checks in 6 sources of blank plasma, matrix effects, recovery of analytes and internal standard through extraction, dilution of over-range samples and stability of analytes in plasma and standard solution. In conclusion, an LC-MS/MS method for the measurement of 13C-LAE, 13C-LAS and 13C-arginine in human plasma has been successfully validated for use.

Acceptable linearity, precision, accuracy and specificity were observed over the concentration range 1 to 200 ng/ml for 13C-LAE and 13C-LAS and over 20 to 1000 ng/ml for 13C-arginine.

 

The low limit of quantification was 1 ng/ml for 13C-LAE, 1 ng/ml for  13C-LAS and 20 ng/ml for 13C -arginine based upon a plasma volume of 400 µl.

 

The recovery of all three analytes and their internal standards from plasma was consistent over the calibration range.

 

There were no interfering peaks from human plasma that affected the measurement of any analyte or internal standard. A peak was presentat the retention time of N-methyl-L-arginine in the chromatograms of blank plasma extracts, but was considered to be insignificant compared to the level of the spiked internal standard.

 

The effect of the variability of the matrix from different humans onthe reliability of the method was shown to be negligible.

 

Plasma samples containing concentrations of an analyte in excess of the validated range could be measured precisely and accurately after a 10-fold dilution with blank plasma to enablemeasurement up to a maximum concentration of 2000 ng/ml for 13C-LAE and 13C-LAS and10000 ng/ml for 13C-arginine using this method.

 

The analytes (13C-LAE,13C-LAS and 13C-arginine) were shown to be stable in plasma whilst on ice (c.a.C) for 2 hours, at nominally -70°C for 31 days and following 3 freeze/thaw cycles. The analytes were also stable in extracted samples at c.a. 22°C (room temperature) for 7 days and atc.a.+4°C for 6 days.

 

In conclusion, an LC-MS/MSmethod for the measurement of 13C-LAE,13C-LAS and 13C-arginine in human plasma has been successfully validated for use.