ASTM E3323-22
(Test Method)Standard Test Method for Lipid Quantitation in Liposomal Formulations Using High Performance Liquid Chromatography (HPLC) with an Evaporative Light-Scattering Detector (ELSD)
Standard Test Method for Lipid Quantitation in Liposomal Formulations Using High Performance Liquid Chromatography (HPLC) with an Evaporative Light-Scattering Detector (ELSD)
SIGNIFICANCE AND USE
5.1 Lipid composition in a liposomal formulation is an important aspect during synthesis of liposomes, which determines stability, surface characteristics, drug encapsulation, and drug release capabilities. The cholesterol component plays a key role in controlled drug release by adding stability to the liposome. A small variation in the lipid composition can significantly alter the parameters mentioned above (15).
5.2 Variation in the lipid composition in the liposomal formulation may influence the safety and efficacy of the product. Therefore, chemical composition of the liposomes shall be determined.
5.3 The pharmaceutical industry and regulatory agencies require QC, QA, specifications, thorough characterization, and quantification of lipid components (16, 17).
5.4 This test method can be used to ascertain variations in the lipid component profiling of various liposomal formulations. However, this test method does not intend to identify chemical degradation products (18).
5.5 Analyzing the stability of analytes and their chemical degradation profiles as a result of oxidation or hydrolysis is beyond the scope of this test method (18, 19).
SCOPE
1.1 This test method describes an analytical technique to quantify lipid components that are often present in liposomal formulations as major components.
1.2 This test method uses high performance liquid chromatography (HPLC) to separate lipids in liposomal formulations and evaporative light-scattering detection (ELSD) to quantify the individual components.
1.3 This test method quantifies three major organic components in liposomal formulations: cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG 2000), and hydrogenated soy L-α-phosphatidylcholine (HSPC).
1.4 This test method can estimate the absolute concentration of cholesterol, DSPE-PEG 2000, and HSPC and their ratio (DSPE-PEG 2000: HSPC: cholesterol) in liposomal formulations.
1.5 This test method describes preparation of calibration standards and samples, HPLC and ELSD instrumentation, method development and method validation, sample analysis, and data reporting.
1.6 The detection limits and quantitation limits for the analytes (lipid components) in this test method are in the range of 2 µg/g to 4 µg/g and 7 µg/g to 10 μg/g, respectively. The analytical measurement ranges for cholesterol, DSPE-PEG 2000, and HSPC are 10 µg/g to 165 µg/g, 10 µg/g to 300 µg/g, and 10 µg/g to 200 µg/g, respectively.
1.7 Significant digits and rounding of all reported values have been performed according to the guidelines as established in Practice D6026.
1.8 Units—The values stated in SI units are to be regarded as the standard. Where appropriate, c.g.s units in addition to SI units are included in this standard.
1.9 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.
1.10 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
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Standards Content (Sample)
This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E3323 − 22
Standard Test Method for
Lipid Quantitation in Liposomal Formulations Using High
Performance Liquid Chromatography (HPLC) with an
1
Evaporative Light-Scattering Detector (ELSD)
This standard is issued under the fixed designation E3323; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 1.9 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
1.1 This test method describes an analytical technique to
responsibility of the user of this standard to establish appro-
quantify lipid components that are often present in liposomal
priate safety, health, and environmental practices and deter-
formulations as major components.
mine the applicability of regulatory limitations prior to use.
1.2 This test method uses high performance liquid chroma-
1.10 This international standard was developed in accor-
tography (HPLC) to separate lipids in liposomal formulations
dance with internationally recognized principles on standard-
and evaporative light-scattering detection (ELSD) to quantify
ization established in the Decision on Principles for the
the individual components.
Development of International Standards, Guides and Recom-
1.3 This test method quantifies three major organic compo-
mendations issued by the World Trade Organization Technical
nentsinliposomalformulations:cholesterol,1,2-distearoyl-sn-
Barriers to Trade (TBT) Committee.
glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (DSPE-PEG 2000), and hydrogenated soy L-α-
2. Referenced Documents
phosphatidylcholine (HSPC).
2
2.1 ASTM Standards:
1.4 Thistestmethodcanestimatetheabsoluteconcentration
D1193Specification for Reagent Water
of cholesterol, DSPE-PEG 2000, and HSPC and their ratio
D1356Terminology Relating to Sampling and Analysis of
(DSPE-PEG 2000: HSPC: cholesterol) in liposomal formula-
Atmospheres
tions.
D6026Practice for Using Significant Digits and Data Re-
1.5 This test method describes preparation of calibration
cords in Geotechnical Data
standards and samples, HPLC and ELSD instrumentation,
D7439Test Method for Determination of Elements in Air-
method development and method validation, sample analysis,
borne Particulate Matter by Inductively Coupled Plasma-
and data reporting.
–Mass Spectrometry
E131Terminology Relating to Molecular Spectroscopy
1.6 The detection limits and quantitation limits for the
E177Practice for Use of the Terms Precision and Bias in
analytes(lipidcomponents)inthistestmethodareintherange
ASTM Test Methods
of 2µg⁄g to 4 µg/g and 7µg⁄g to 10 µg/g, respectively. The
E456Terminology Relating to Quality and Statistics
analytical measurement ranges for cholesterol, DSPE-PEG
E682Practice for Liquid Chromatography Terms and Rela-
2000, and HSPC are 10µg⁄g to 165 µg/g, 10µg⁄g to 300 µg/g,
tionships
and 10µg⁄g to 200 µg/g, respectively.
E2490Guide for Measurement of Particle Size Distribution
1.7 Significant digits and rounding of all reported values
of Nanomaterials in Suspension by Photon Correlation
havebeenperformedaccordingtotheguidelinesasestablished
Spectroscopy (PCS)
in Practice D6026.
E3025Guide for TieredApproach to Detection and Charac-
1.8 Units—The values stated in SI units are to be regarded
terization of Silver Nanomaterials in Textiles
asthestandard.Whereappropriate,c.g.sunitsinadditiontoSI
E3080Practice for Regression Analysis with a Single Pre-
units are included in this standard.
dictor Variable
1
This test method is under the jurisdiction of ASTM Committee E56 on
Nanotechnology and is the direct responsibility of Subcommittee E56.08 on
2
Nano-Enabled Medical Products. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved Oct. 1, 2022. Published November 2022. Originally contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
approved in 2021. Last previous edition approved in 2021 as E3323–21. DOI: Standards volume information, refer to the standard’s Document Summary page on
10.1520/E3323-22. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
---------------------- Page: 1 ----------------------
E3323 − 22
t
2
3. Terminology
A~t! 5 * ~S!dt (1)
t
1
3.1 Definitions:
Where A(t) and S(t) = Peak area and the instantaneous
3.1.1 accuracy, n—closeness of agreement between a test
detector’s response at time, t, res
...
This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: E3323 − 21 E3323 − 22
Standard Test Method for
Lipid Quantitation in Liposomal Formulations Using High
Performance Liquid Chromatography (HPLC) with an
1
Evaporative Light-Scattering Detector (ELSD)
This standard is issued under the fixed designation E3323; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope
1.1 This test method describes an analytical technique to quantify lipid components that are often present in liposomal
formulations as major components.
1.2 This test method uses high performance liquid chromatography (HPLC) to separate lipids in liposomal formulations and
evaporative light-scattering detection (ELSD) to quantify the individual components.
1.3 This test method quantifies three major organic components in liposomal formulations: cholesterol, 1,2-distearoyl-sn-glycero-
3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG 2000), and hydrogenated soy L-α-
phosphatidylcholine (HSPC).
1.4 This test method can estimate the absolute concentration of cholesterol, DSPE-PEG 2000, and HSPC and their ratio
(DSPE-PEG 2000: HSPC: cholesterol) in liposomal formulations.
1.5 This test method describes preparation of calibration standards and samples, HPLC and ELSD instrumentation, method
development and method validation, sample analysis, and data reporting.
1.6 The detection limits and quantitation limits for the analytes (lipid components) in this test method are in the range of 22 μg ⁄g
to 4 μg/g and 77 μg ⁄g to 10 μg/g, respectively. The analytical measurement ranges for cholesterol, DSPE-PEG 2000, and HSPC
are 1010 μg ⁄g to 165 μg/g, 1010 μg ⁄g to 300 μg/g, and 1010 μg ⁄g to 200 μg/g, respectively.
1.7 Significant digits and rounding of all reported values have been performed according to the guidelines as established in
Practice D6026.
1.8 Units—The values stated in SI units are to be regarded as the standard. Where appropriate, c.g.s units in addition to SI units
are included in this standard.
1.9 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of
regulatory limitations prior to use.
1
This test method is under the jurisdiction of ASTM Committee E56 on Nanotechnology and is the direct responsibility of Subcommittee E56.08 on Nano-Enabled
Medical Products.
Current edition approved Dec. 15, 2021Oct. 1, 2022. Published April 2022November 2022. Originally approved in 2021. Last previous edition approved in 2021 as
E3323 – 21. DOI: 10.1520/E3323-21.10.1520/E3323-22.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1
---------------------- Page: 1 ----------------------
E3323 − 22
1.10 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2
2.1 ASTM Standards:
D1193 Specification for Reagent Water
D1356 Terminology Relating to Sampling and Analysis of Atmospheres
D6026 Practice for Using Significant Digits and Data Records in Geotechnical Data
D7439 Test Method for Determination of Elements in Airborne Particulate Matter by Inductively Coupled Plasma–Mass
Spectrometry
E131 Terminology Relating to Molecular Spectroscopy
E177 Practice for Use of the Terms Precision and Bias in ASTM Test Methods
E456 Terminology Relating to Quality and Statistics
E682 Practice for Liquid Chromatography Terms and Relationships
E2490 Guide for Measurement of Particle Size Distribution of Nanomaterials in Suspension by Photon Correlation Spectroscopy
(PCS)
E3025 Guide for Tiered Approach to Detection and Characterization of Silver Nanomaterials in Textiles
E3080 Practice for Regression Analysis with a Single Predictor Variable
3. Terminology
3.1 Definitions:
3.1.1 accuracy, n—closeness of agreement between a test result and an accepted reference value.
3.1.1.1 Discussion—
The term accuracy, when applied to a set of results, involves a combination of ran
...
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