ASTM E3297-21

Standard Test Method for Lipid Quantitation in Liposomal Formulations Using High Performance Liquid Chromatography (HPLC) with a Charged Aerosol Detector (CAD)

SIGNIFICANCE AND USE
5.1 The growing interest in liposomal formulations in the pharmaceutical industry requires QC and thorough characterization and quantification of lipids that form liposomes (6). Lipid composition has proven to be a critical attribute of the liposomal formulation; it directly influences the stability of the formulation, drug loading, performance, size, and surface characteristics of the liposome. Cholesterol plays a key role in controlled drug release by adding stability to the liposome (7). Significant variation in the lipid composition and ratio of the components will influence the safety, biodistribution, drug efficacy, and drug release kinetics of the liposomal formulation (8-11).  
5.2 This test method is a fast and reliable procedure for the quantification of cholesterol, DSPE-PEG 2000, and HSPC in liposomal formulations using HPLC-CAD.  
5.3 This test method can be used for QC and QA and to ascertain variations in component profiling of liposomal formulations.
SCOPE
1.1 This test method is for the separation of lipids in liposomal formulations through high performance liquid chromatography (HPLC) and their quantitation using a mass-flow sensitive charged aerosol detector (CAD).  
1.2 This test method is specifically for liposomal formulations containing cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG 2000) and hydrogenated soy L-α-phosphatidylcholine (HSPC).  
1.3 This test method is applicable to report the absolute concentrations and ratio of cholesterol, DSPE-PEG 2000, and HSPC in liposomal formulations. Assessment of the stability of the analytes in terms of their degradation profiles as a result of oxidation or hydrolysis is beyond the scope of this test method.  
1.4 This test method includes calibration standards preparation, sample preparation, method validation, and sample analysis. This method also contains specifications for instrumentation and the chromatography experimental procedure.  
1.5 The detection limit and quantitation limit for the analytes in this test method is in the range of 0.1–2.0 µg/g and 1.0–5.0 μg/g respectively. The analytical measurement range for cholesterol, DSPE-PEG 2000, and HSPC is 5–300 µg/g.  
1.6 All observed and calculated values shall conform to the guidelines for significant digits and rounding as established in Practice D6026.  
1.7 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.  
1.8 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.  
1.9 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.

General Information

• Status: Published
• Publication Date: 14-Dec-2021
• ICS: 07.120 - Nanotechnologies
• Technical Committee: E56 - Nanotechnology
• Drafting Committee: E56.08 - Nano-Enabled Medical Products

Relations

• Refers: ASTM E3080-23 - Standard Practice for Regression Analysis with a Single Predictor Variable
• Effective Date: 01-Nov-2023
• Refers: ASTM E456-13a(2022)e1 - Standard Terminology Relating to Quality and Statistics
• Effective Date: 01-Apr-2022
• Refers: ASTM D1356-20a - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 01-Sep-2020
• Refers: ASTM D1356-20 - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 15-Mar-2020
• Refers: ASTM E3080-19 - Standard Practice for Regression Analysis with a Single Predictor Variable
• Effective Date: 01-Sep-2019
• Refers: ASTM E682-92(2019) - Standard Practice for Liquid Chromatography Terms and Relationships
• Effective Date: 01-Sep-2019
• Refers: ASTM E3080-17 - Standard Practice for Regression Analysis
• Effective Date: 01-Nov-2017
• Refers: ASTM E456-13A(2017)e3 - Standard Terminology Relating to Quality and Statistics
• Effective Date: 01-Oct-2017
• Refers: ASTM E456-13A(2017)e1 - Standard Terminology Relating to Quality and Statistics
• Effective Date: 01-Oct-2017
• Refers: ASTM E3080-16 - Standard Practice for Regression Analysis
• Effective Date: 01-Nov-2016
• Refers: ASTM D1356-15a - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 15-Oct-2015
• Refers: ASTM D1356-15 - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 01-Jul-2015
• Refers: ASTM D1356-14b - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 01-Dec-2014
• Refers: ASTM E177-14 - Standard Practice for Use of the Terms Precision and Bias in ASTM Test Methods
• Effective Date: 01-May-2014
• Refers: ASTM D1356-14a - Standard Terminology Relating to Sampling and Analysis of Atmospheres
• Effective Date: 01-May-2014

Overview

ASTM E3297-21 is an international test method developed by ASTM for the quantification of lipids in liposomal formulations using high performance liquid chromatography (HPLC) combined with a charged aerosol detector (CAD). With the increasing use of liposomal drug delivery systems in the pharmaceutical sector, accurate determination of lipid composition is essential for ensuring product stability, efficacy, and safety. This standard provides a validated and reliable procedure for measuring key lipids-cholesterol, DSPE-PEG 2000, and HSPC-in liposomal preparations, supporting both quality control (QC) and quality assurance (QA) efforts.

Key Topics

• Lipid Quantitation in Liposomal Formulations
  • Designed for liposomal products containing cholesterol, DSPE-PEG 2000, and hydrogenated soy L-α-phosphatidylcholine (HSPC)
  • Focuses on separation and quantification using HPLC with CAD, enabling mass-flow sensitive detection of analytes
• Scope of the Test Method
  • Reports absolute concentrations and component ratios of the three target lipids
  • Includes calibration standards preparation, method validation, and sample analysis procedures
  • Specifies performance criteria such as detection and quantitation limits, and measurement ranges for the analytes
  • Does not cover degradation or stability assessment of analytes
• Analytical Validation
  • Test method validation covers linearity, accuracy, precision, and specificity
  • Requires careful handling and storage of samples and calibration standards to prevent degradation
• Quality Control Applications
  • Enables monitoring and verification of lipid profiles during formulation and release testing
  • Supports component profiling to ensure product consistency and performance

Applications

ASTM E3297-21 is practically valuable for laboratories, manufacturers, and regulatory professionals in the pharmaceutical and biotechnology fields. Its main applications include:

• Pharmaceutical Quality Control and Assurance
  • Used to verify the consistency of lipid content in liposomal drug formulations
  • Supports batch release testing by ensuring conformity with product specifications
• Product Development and Optimization
  • Facilitates thorough characterization of liposomal components during research, formulation development, and process optimization
• Regulatory Submissions
  • Provides standardized, reproducible results that aid in regulatory filings and demonstrate compliance with international guidelines
• Troubleshooting and Process Monitoring
  • Detects variations in lipid ratios that could impact drug release, bioavailability, or stability
  • Assists in identifying sources of batch variability and optimizing manufacturing conditions

Related Standards

• ASTM D1193: Specification for Reagent Water
• ASTM D6026: Practice for Using Significant Digits and Data Records in Geotechnical Data
• ASTM E131: Terminology Relating to Molecular Spectroscopy
• ASTM E682: Practice for Liquid Chromatography Terms and Relationships
• ASTM E2490: Guide for Measurement of Particle Size Distribution of Nanomaterials in Suspension
• ASTM E3080: Practice for Regression Analysis with a Single Predictor Variable

These related ASTM standards provide foundational terminology, water quality, statistical practices, and chromatographic methodology essential for implementing and understanding E3297-21.

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Keywords: ASTM E3297-21, lipid quantitation, liposomal formulation, HPLC-CAD, pharmaceutical QC, cholesterol, DSPE-PEG 2000, HSPC, quality assurance, analytical validation, liposome standard, component profiling, drug delivery characterization.

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: E3297 − 21
Standard Test Method for
Lipid Quantitation in Liposomal Formulations Using High
Performance Liquid Chromatography (HPLC) with a
Charged Aerosol Detector (CAD)
This standard is issued under the fixed designation E3297; 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 priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
1.1 This test method is for the separation of lipids in
1.9 This international standard was developed in accor-
liposomal formulations through high performance liquid chro-
dance with internationally recognized principles on standard-
matography (HPLC) and their quantitation using a mass-flow
ization established in the Decision on Principles for the
sensitive charged aerosol detector (CAD).
Development of International Standards, Guides and Recom-
1.2 This test method is specifically for liposomal formula-
mendations issued by the World Trade Organization Technical
tions containing cholesterol, 1,2-distearoyl-sn-glycero-3-
Barriers to Trade (TBT) Committee.
phosphoethanolamine-N-[methoxy(polyethylene glycol)-
2. Referenced Documents
2000] (DSPE-PEG 2000) and hydrogenated soy L-α-
phosphatidylcholine (HSPC).
2.1 ASTM Standards:
D1193Specification for Reagent Water
1.3 This test method is applicable to report the absolute
concentrations and ratio of cholesterol, DSPE-PEG 2000, and D1356Terminology Relating to Sampling and Analysis of
Atmospheres
HSPCinliposomalformulations.Assessmentofthestabilityof
the analytes in terms of their degradation profiles as a result of D6026Practice for Using Significant Digits and Data Re-
cords in Geotechnical Data
oxidationorhydrolysisisbeyondthescopeofthistestmethod.
D7439Test Method for Determination of Elements in Air-
1.4 This test method includes calibration standards
borne Particulate Matter by Inductively Coupled Plasma-
preparation, sample preparation, method validation, and
–Mass Spectrometry
sample analysis. This method also contains specifications for
E131Terminology Relating to Molecular Spectroscopy
instrumentation and the chromatography experimental proce-
E177Practice for Use of the Terms Precision and Bias in
dure.
ASTM Test Methods
1.5 The detection limit and quantitation limit for the ana-
E456Terminology Relating to Quality and Statistics
lytes in this test method is in the range of 0.1–2.0 µg/g and
E682Practice for Liquid Chromatography Terms and Rela-
1.0–5.0 µg/g respectively. The analytical measurement range
tionships
for cholesterol, DSPE-PEG 2000, and HSPC is 5–300 µg/g.
E2490Guide for Measurement of Particle Size Distribution
of Nanomaterials in Suspension by Photon Correlation
1.6 All observed and calculated values shall conform to the
Spectroscopy (PCS)
guidelines for significant digits and rounding as established in
E3025Guide for TieredApproach to Detection and Charac-
Practice D6026.
terization of Silver Nanomaterials in Textiles
1.7 Units—The values stated in SI units are to be regarded
E3080Practice for Regression Analysis with a Single Pre-
asthestandard.Whereappropriate,c.g.sunitsinadditiontoSI
dictor Variable
units are included in this standard.
3. Terminology
1.8 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
3.1 Definitions:
responsibility of the user of this standard to establish appro-
3.1.1 accuracy, n—closeness of agreement between a test
result and an accepted reference value.
This test method is under the jurisdiction of ASTM Committee E56 on
Nanotechnology and is the direct responsibility of Subcommittee E56.08 on For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Nano-Enabled Medical Products. contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
Current edition approved Dec. 15, 2021. Published April 2022. DOI: 10.1520/ Standards volume information, refer to the standard’s Document Summary page on
E3297-21. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3297 − 21
n 2
3.1.1.1 Discussion—The term accuracy, when applied to a
X Y
S D
( i i
set of results, involves a combination of random components
i51
r 5 (2)
n n
and a common systematic error or bias component. E177
X Y
S DS D
( i ( i
3.1.2 aerosol, n—suspension of solid particles or liquid i51 i51
droplets or both in a gaseous medium. D1356
Where n = number of observations. E131
3.1.3 analyte, n—chemical constituent of interest in an
3.1.12 intermediate precision, n—closeness of agreement
analytical procedure. E3025
between test results obtained under specified intermediate
3.1.4 analytical instrument qualification, n—collection of
precision conditions. E177
documented evidence that an instrument performs suitably for
3.1.13 intermediate precision conditions, n—conditions un-
its intended purpose (1).
der which test results are obtained with the same test method
3.1.5 baseline noise, n—combinationofhigh-frequencysig-
using test units taken at random from a single quantity of
nal fluctuations and low-frequency signal drift that affect
material that is as nearly homogeneous as possible and with
baseline stability.
changing conditions such as operator, measuring equipment,
3.1.5.1 Discussion—These signal fluctuations can originate
location within the laboratory, and time. E177
from line-voltage fluctuations, shot noise (Poisson noise) from
3.1.14 linearity, n—ability of the analytical method (within
electronic circuits, improper solvent degassing, temperature
a certain range) to obtain test results that are directly propor-
instability,andothernonequilibriumeffects.Itisrepresentative
tional to the concentration (amount) of the analyte in the
of detector response that is not related to responses from
sample (3).
analytes or matrix interferences.
3.1.15 lipids, n—diverse group of organic compounds that
3.1.6 calibration curve, n—relationship between measured
are soluble in organic solvents but are insoluble in water.
response values and analytical concentrations of a standard or
3.1.15.1 Discussion—In this test method, lipids refer to
reference material. D7439
cholesterol, DSPE-PEG 2000, and HSPC. The chemical struc-
3.1.6.1 Discussion—A set of calibration standards are used
tures of these three lipids are presented in Appendix X1.
to construct a calibration curve, and the concentration of
analyte present in an unknown sample can be determined by
3.1.16 liposomal formulation, n—product designed to assist
comparing the detector response with the calibration curve.
in the delivery of an active pharmaceutical ingredient, either
encapsulated or intercalated in a liposome.
3.1.7 calibration standards, n—set of solutions with known
3.1.16.1 Discussion—Formulated products can contain
analyte concentration used to construct calibration curves.
vesicles having a single lipid bilayer (unilamellar), multiple
3.1.8 carryover effect, n—systematic error that is derived
concentric lipid bilayers (multilamellar) or a mixture of unila-
from a component from the preceding sample injection being
mellar and multilamellar vesicles.
introducedintothenextsampleaffectingaccuratequantitation.
3.1.17 liposome, n—synthetic vesicle composed of a one or
3.1.9 cholesterol, n—steroidal organic compound that stabi-
more bilayers formed by amphipathic molecules such as
lizes the lipid bilayer in liposomal formulations.
phospholipids that enclose a central aqueous compartment.
3.1.10 chromatogram, n—graphical presentation of detector
Adapted from (4).
response plotted as a function of elution time or effluent
3.1.18 matrix blank, n—substance that closely matches the
volume as the sample components elute from the column and
samples being analyzed with regard to matrix components, but
reach the detector.
has none of the analytes of interest.
3.1.10.1 Discussion—In this test method, the charged aero-
3.1.18.1 Discussion—Ideally, the matrix blank does not
sol detector (CAD) response expressed as current (pA) is
contain the analyte(s) of interest but is subjected to all
plotted against elution time (min). For analysis, the character-
sample-processing operations including all reagents used to
istic detector response of the eluting analyte is typically
analyzethetestsamples.Thematrixblankisusedtodetermine
evaluated from the peak area recorded in the chromatogram.
the absence of significant interference as a result of the matrix,
This peak area (A) can be expressed mathematically as the
reagents, and equipment used in the analysis.
integral of detector response for analyte over an elution time
interval from t to t :
1 2
3.1.19 matrix effect, n—influence of one or more compo-
t
2 nents from the sample matrix on the measurement of the
A~t! 5 ~S!t (1)
*
t
1 analyte concentration or mass.
Where A(t) and S(t) = Peak area and the instantaneous 3.1.19.1 Discussion—Matrix effects may be observed as
enhanced or suppressed detector responses compared with
detector’s response at time, t, respectively (2).
those produced by simple solvent solutions of the analyte (5).
3.1.11 coeffıcient of determination, n—statisticalmeasureof
the linear relationship between X and Y calculated by:
3.1.20 mobile phase, n—solvent used to sweep or elute the
sample components through the column that may consist of a
single component or a mixture of components.
3.1.20.1 Discussion—The term eluent is often used for the
The boldface numbers in parentheses refer to a list of references at the end of
this standard. preferred mobile phase. E682
E3297 − 21
3.1.21 peak area, n—area under the peak obtained from 3.2 Definitions of Terms Specific to This Standard:
integrationofthedetectorsignalabovethebaselineforagiven
3.2.1 test sample, n—final form of the sample that is used
component.
for test determination.
3.2.1.1 Discussion—In this test method, a test unit solubi-
3.1.22 3.1.22 peak resolution (R ), n—measureofchromato-
s
graphic separation of two components in a mixture calculated lized with methanol and diluted to within the bracketed range
is defined as test sample.
by:
t 2 t
~ ! 3.2.2 test unit, n—portion of a material that is sufficient to
R2 R1
R 5 2 3 (3)
s
~w 1 w ! acquire test result(s) for the property(-ies) to be measured.
1 2
3.2.2.1 Discussion—In this test method, the original lipo-
where:
somal formulation to be tested for lipid quantitation is defined
t and t = retention time of the two components 1 and 2
R2 R1
as a test unit.
(t > t ), and
R2 R1
w and w = corresponding widths at the bases of the peaks 3.3 Acronyms:
1 2
obtainedbyextrapolatingtherelativelystraight
3.3.1 CAD—Charged aerosol detector
sides of the peaks to the baseline.
3.3.2 CRM—Certified reference material
3.1.23 precision, n—closeness of agreement between inde-
3.3.3 DSPE-PEG—1,2-distearoyl-sn-glycero-3-phospho-
pendent test results obtained under stipulated conditions. E177
ethanolamine-N-[methoxy(polyethylene glycol)-2000]
3.1.24 regression analysis, n—statistical procedure used to
3.3.4 HPLC—High performance liquid chromatography
characterize the association between two or more numerical
variables for prediction of the response variable from the
3.3.5 HSPC—Hydrogenated soy L-α-phosphatidylcholine
predictor variable.
3.3.6 ID—Inside diameter
3.1.24.1 Discussion—Theobjectiveistoobtainaregression
3.3.7 LC-MS—Liquid chromatography-mass spectrometry
model for use in predicting the value of the response variable
for given values of the predictor variable. In this test method,
3.3.8 LOD—Limit of detection
the predictor variable is CAD signal (pA) and the response
3.3.9 LOQ—Limit of quantitation
variable is mass concentration. E3080
3.3.10 QA—Quality assurance
3.1.25 repeatability, n—precision of test results from tests
conductedwithintheshortestpracticaltimeperiodonidentical
3.3.11 QC—Quality control
materialbythesametestmethodinasinglelaboratorywithall
3.3.12 RCF—Relative centrifugal force
known sources of variable conditions controlled at the same
levels. Adapted from E177. 3.3.13 RSD—Relative standard deviation
3.1.26 reproducibility, n—precisionoftestresultsfromtests 3.3.14 SD—Standard deviation
conducted on identical material by the same test method in
3.3.15 µg/g—Parts per million
different laboratories with different operators using different
3.3.16 UHPLC—Ultra high performance liquid chromatog-
equipment. Adapted from E456.
raphy
3.1.27 robustness, n—measure of change in the outcome of
an analytical procedure with deliberate and systematic varia-
4. Summary of Test Method
tions in any or all of the key method parameters that influence
it. Adapted from E2490.
4.1 Cholesterol, DSPE-PEG 2000, and HSPC in the lipo-
somalformulationaresolubilizedinmethanolat1:100dilution
3.1.28 solvent blank, n—solution containing all reagents
byvolumefollowedbyvortexmixing.Thissolubilizedsample
used in sample dissolution in the same quantities used for
is further subjected to quantitative analysis using reversed-
preparation of blank and sample solutions. D7439
phase HPLC with CAD. Before sample analysis, the test
3.1.28.1 Discussion—The solvent blank is used to assess
method is validated for linearity, precision, accuracy,
contamination from the laboratory environment and character-
specificity, LOD, and LOQ.
ize spectral background from the reagents used in sample
4.2 Calibration curves of concentrations ranging from
preparation.
5–300 µg/g are established for cholesterol, DSPE-PEG 2000,
3.1.29 specificity, n—ability to assess unequivocally the
and HSPC. For analysis, as CAD response is nonlinear in the
analyte in the presence of components that may be expected to
measured range, the linear regression model is applicable only
be present.
afterlogarithmictransformation.Hence,log(peakarea)versus
3.1.29.1 Discussion—Typically, these might include
log (concentration) is plotted for each analyte to obtain the
impurities, degradants, matrix, and so forth (3).
corresponding calibration curve. Linear regression analysis
3.1.30 system suitability, n—determination of instrument givestheslopeandinterceptsnecessarytoquantifycholesterol,
performance in a particular procedure (for example, sensitivity DSPE-PEG 2000, and HSPC in the test samples. Lipid
and chromatographic retention) by analyzing a set of appro- component ratio (DSPE-PEG 2000: HSPC:cholesterol) shall
priate reference standards before the analytical run. be reported along with total lipid content in milligrams/grams.
E3297 − 21
5. Significance and Use 7.3 Reversed-phase HPLC BEH (Bridged Ethylene Hybrid)
column-C18,13nmporesize,3.5µmparticlesize,3mm×150
5.1 The growing interest in liposomal formulations in the
mm (ID × length) or equivalent column that can resolve all
pharmaceutical industry requires QC and thorough character-
analytes of interest with R ≥ 1.5.
s
ization and quantification of lipids that form liposomes (6).
Lipid composition has proven to be a critical attribute of the 7.4 Analytical balance that can accurately weigh with read-
ability up to 0.0001 g.
liposomal formulation; it directly influences the stability of the
formulation, drug loading, performance, size, and surface
7.5 Vortex mixer.
characteristics of the liposome. Cholesterol plays a key role in
7.6 Mechanical pipettes with disposable tips covering from
controlled drug release by adding stability to the liposome (7).
0.002 to 10 mL.
Significant variation in the lipid composition and ratio of the
components will influence the safety, biodistribution, drug
7.7 Solvent reservoir bottles, 1 L.
efficacy,anddrugreleasekineticsoftheliposomalformulation
7.8 Volumetric flask, 10 mL and 1 L.
(8-11).
7.9 Measuring cylinder, 0.5 and 1 L.
5.2 This test method is a fast and reliable procedure for the
7.10 Polypropylene tubes, 15 mL.
quantification of cholesterol, DSPE-PEG 2000, and HSPC in
liposomal formulations using HPLC-CAD.
7.11 Glass amber vials, 5, 10, and 20 mL.
5.3 This test method can be used for QC and QA and to
7.12 Autosampler amber vials, 2 mL.
ascertain variations in component profiling of liposomal for-
7.13 Ultrasonic bath.
mulations.
7.14 0.2 µm bottle top vacuum filter.
6. Interferences
8. Reagents and Materials
6.1 Interferences caused by solvent impurities could lead to
8.1 Purity of Reagents—Reagent-grade chemicals shall be
high baseline noise; hence, in this test method, LC-MS grade
used in all tests. Unless otherwise indicated, it is intended that
solvents are recommended.
all reagents conform to the specifications of the Committee on
6.2 Useahigh-qualitynitrogensource(≥95%purity)thatis
Analytical Reagents of theAmerican Chemical Society where
free from water vapor, particles, and nonvolatile hydrocarbons 4
such specifications are available. Other grades may be used,
such as compressor oils. The use of gases that allow either
provided it is first ascertained that the reagent is of sufficiently
combustion of solvents or oxidation of analytes should be
high purity to permit its use without lessening the accuracy of
avoided.
the determination.
8.1.1 Acetonitrile, LC-MS grade.
6.3 Keep the auto sampler injection port and column clean
8.1.2 Methanol, LC-MS grade.
as per the manufacturer’s recommendations to avoid carryover
orghostpeaks.Contaminationofglasscontainersorvialsused 8.1.3 Ammonium acetate deliquescent crystals, LC-MS
grade.
for this test method should be avoided.
8.1.4 Purity of Water—Unless otherwise indicated, refer-
6.4 If the user observes any matrix effects under the
ences to water shall be understood to mean reagent water as
recommended test conditions, optimization of the sample
defined byType 1 of Specification D1193. Use deionized (>18
preparation or modification in the chromatographic parameters
MΩ cm) high-purity (Type 1) water.
(for example, solvent gradient or eluent flow rate) may be
required. The Bligh-Dyer method described in Appendix X3 8.2 Materials:
8.2.1 Cholesterol, >99 % purity, powder form.
could potentially be adopted to remove water soluble excipi-
ents in test liposomal formulations if deemed necessary. 8.2.2 HSPC, >99 % purity, consisting of C16:0 (HSPC 1)
and C18:0 (HSPC 2) fatty acids, powder form.
6.5 Allthestocksolutions,calibrationstandards(thatis,six
8.2.3 DSPE-PEG 2000, >99 % purity, powder form.
calibration levels) and the test samples should be stored either
at 0–4°C or –20°C as recommended in this test method to
9. Hazards
avoid degradation of target analytes.
9.1 Proper protective measures are required while handling
6.6 Chemicals with high purity shall be used for the
liposomal formulations. This test method uses methanol and
preparation of lipid calibration standards. When feasible, it is
acetonitrile, which are flammable. All solvents should be
recommended that higher-order reference standards (for
handled in a chemical fume hood to avoid inhalation. Organic
example, CRMs) be acquired for calibration standards. If
referencematerialsarenotavailable,high-qualitycrystallineor
lyophilized chemicals of known purity can be used.
ACS Reagent Chemicals, Specifications and Procedures for Reagents and
Standard-Grade Reference Materials, American Chemical Society, Washington,
7. Apparatus
DC. For suggestions on the testing of reagents not listed by theAmerican Chemical
Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset,
7.1 HPLC with in-line degasser module.
U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharma-
7.2 CAD. copeial Convention, Inc. (USPC), Rockville, MD.
E3297 − 21
turer’s recommendation should be allowed to equilibrate at ambient
solvent waste should be disposed of according to local regu-
temperature before weighing.
lations. The CAD outlet should be connected to an exhaust as
per the manufacturer’s recommendation to avoid aerosol ex-
11.1 Deionized(>18MΩcm)watermustbefilteredthrough
posure. a0.2µmbottletopvacuumfilter.Thisstepisnotneededinthe
case of a water purification system with an attached 0.2 µm
10. Mobile Phase Preparation
filter.
10.1 Mobile Phase A: Acetonitrile/Water (90/10 v/v) + 5
11.2 Preparation of Primary Stock Solutions:
mmol/L Ammonium Acetate:
11.2.1 Rinse three empty 20 mL glass amber vials with
10.1.1 Deionized (>18 MΩ cm) water must be filtered
deionized water and dry thoroughly. Cap the vials.
through a 0.2 µm bottle top vacuum filter. This step is not
11.2.2 Label each vial with the corresponding component
needed in the case of a water purification system with an
(that is, analyte) j, where j = cholesterol, DSPE-PEG 2000, or
attached 0.2 µm filter.
HSPC.
10.1.2 Rinse an empty solvent reservoir bottle (1 L) with
11.2.3 Weigh each capped vial on an analytical balance and
deionized water thoroughly and dry.
record the mass to 62mgas W .
0j
10.1.3 Weigh 3.85 6 0.02 g of LC-MS-grade ammonium
11.2.4 Individually weigh 10 6 2 mg of each component
acetatedeliquescentcrystalsandtransfertoanemptyvolumet-
(cholesterol, DSPE-PEG 2000, and HSPC) on an analytical
ric flask (1 L).
balance.Recordthemassesas W .Transferthecomponentsto
1j
10.1.4 Add ≈800mLof0.2-µmfiltereddeionizedwaterand
the appropriately labeled vials.
dissolve the salt thoroughly. Then, fill the volumetric flask up
11.2.5 Rinse an empty volumetric flask with methanol and
to the 1L graduation mark with deionized water to make a
dry thoroughly.
homogenous 50 mmol/L solution of ammonium acetate.
11.2.6 Measure 10 mL of LC-MS-grade methanol in the
10.1.5 Toprepare1LofmobilephaseA,mix100mLof50
clean volumetric flask. Add to the vial from 11.2.4labeled
mmol/L ammonium acetate and 900 mL of LC-MS-grade
cholesterol. Cap the vial.
acetonitrile in the reservoir bottle (1 L) using a measuring
11.2.7 Repeat 11.2.5 and 11.2.6 for DSPE-PEG 2000 and
cylinder.
HSPC.
10.1.6 DegasmobilephaseAfor10minusinganultrasonic
11.2.8 Weigh each capped vial on an analytical balance and
bath at atmospheric pressure. Use of an in-line degasser
record the mass to 62mgas W .
2j
module further helps to achieve a stable baseline during an
11.2.9 Dissolve the solids in each vial thoroughly by vortex
analytical run.
mixing for 1 min followed by bath sonication for 5 min.
10.2 Mobile Phase B: Methanol + 5 mmol/L Ammonium
11.2.10 Store the vials of the individual stock solutions at
Acetate:
–20°C until needed. Primary stock solutions are stable up to
10.2.1 To prepare 1 L of mobile phase B, weigh 385 6 10
four months under this condition.
mg of LC-MS-grade ammonium acetate deliquescent crystals
11.2.11 The individual stock concentrations of the three
and transfer it to an empty volumetric flask (1 L).
components C, are calculated using Eq 4. The purity (j) % for
j
10.2.2 Add ≈500 mLLC-MS-grade methanol to a volumet-
each component should be the value from the manufacturer’s
ricflaskanddissolvethesaltthoroughly.Then,filltheflaskup
certificate of analysis:
to the graduated mark with LC-MS-grade methanol.
Massoftheanalyte w
1j
10.2.3 Transfer all contents to a clean and dry solvent
C 5 5 purity j % 3
~ ~ ! ! S D
j
Totalmassofthesolution w 2 w
2j 0j
reservoir bottle.
10.2.4 DegasmobilephaseBfor10minusinganultrasonic 310 µg/g (4)
bath at atmospheric pressure. Use of an in-line degasser
Where j = cholesterol, DSPE-PEG 2000 or HSPC.
module further helps to achieve a stable baseline during an
11.3 Preparation of Intermediate Stock Solutions:
analytical run.
11.3.1 Rinse three empty 20 mL glass amber vials with
NOTE 1—Sparging with helium can be used as an alternative to the
deionized water and dry thoroughly.
ultrasonic and in-line vacuum degassing combination as recommended in
11.3.2 Label each vial with the corresponding component
this test method.
(analyte), that is, j = cholesterol, DSPE-PEG 2000, or HSPC.
11. Preparation of Calibration Standards
11.3.3 Rinse three empty volumetric flasks with deionized
NOTE 2—Gravimetric Measurements—All working standards in this
water and dry thoroughly.
test method are prepared gravimetrically using an analytical balance
11.3.4 Follow11.2.5–11.2.11(withlesseramountof W to
(0.0001 g accuracy). Although the volumetric preparation shows close 1j
achieve a target concentration C = 500 µg/g).
agreement with the gravimetric preparation, it is known that a (1 to 5) %
j
errorcanbeintroducedduringsmallvolumetransfersand,hence,biasthe
11.3.5 Store the vials of the intermediate stock (I-stock)
quantitation results. The analytical balance provides better measurement
solutions at –20°C until needed. Intermediate stock solutions
resolution (that is, more significant figures) than mechanical pipettes and
are stable up to four months under this condition.
offers better accuracy (12).
NOTE 3—Conditioning the pipette tip with appropriate solvents before
11.4 Calibration Curve:
transfer of calibration standards for weighing, working promptly with the
11.4.1 The calibration curve shall have six levels with
stock solutions, and weighing the volatile liquids in securely capped
analyte concentrations ranging from 5 (Level 1) to 300 (Level
containersusingasecondarycontainerarehighlyrecommendedpractices.
NOTE 4—Chemicals stored in the freezer at –20ºC or as per manufac- 6) µg/g (specifically 5, 25, 50, 100, 150, and 300 µg/g).
E3297 − 21
(r ≥ 0.995). The user is not limited to example target concentrations
11.4.2 Each calibration standard (level) is obtained by
recommended in this test method and any six concentrations (evenly
mixing either the stock solutions or the I-stock solutions of the
spaced) can be used to establish a linear bracketed concentration range
three analytes in a 1:1:1 mass ratio with methanol in a 20 mL
that satisfies the above quality criterion.
vial. Table 1 shows the estimated volumes of each analyte
stock solution or I-stock solution and methanol for the various
12. Preparation of QC, Accuracy, and Precision Samples
calibration levels. Use a gravimetric method similar to that in
12.1 A QC sample should be prepared similarly to calibra-
11.2 and 11.3 to obtain the desired analyte concentrations.
tion standards (11.4.3) by mixing cholesterol, DSPE-PEG
11.4.3 Preparation of Calibration Standards of Analyte
2000, and HSPC stock solutions with a final concentration of
Mixtures:
75 µg/g of each analyte. Mixtures of 75 µg/g will serve as the
11.4.3.1 Rinse one empty 10 mL glass amber vial with
QC sample.
deionized water and dry thoroughly.
11.4.3.2 Rinse one empty volumetric flask with deionized
12.2 Mixtures of 25 µg/g (low), 75 µg/g (medium), and 150
water and dry thoroughly.
µg/g (high) should be prepared for precision and accuracy
11.4.3.3 Start with calibration Level 1 (target concentration
studies.The final concentration calculation shall use gravimet-
= 5 µg/g). Label the 10 mL vial as Level 1.
ric measurements as described in 11.2.
11.4.3.4 Addappropriatevolumesofeachofthethreestock
12.3 To assess the matrix effect, test samples shall contain
solutions (cholesterol, DSPE-PEG 2000, and HSPC) and
all possible matrix components from the test unit. The ratio of
methanol according to Table 1.
concentration of matrix components to analytes in the test
11.4.3.5 Recordthemassusingananalyticalbalanceineach
sample should be close to that of the test unit. The three test
step; mass of the vial with cap (A , g), mass of the vial + each
samples should contain all three analytes, each with a concen-
stocksolutionofanalyte jadded(= A ,g),andmassofthevial
1j
tration of 25 µg/g (low), 75 µg/g (medium), and 150 µg/g
+ stock solutions + methanol (= A , g).
(high).
11.4.3.6 The final concentration of the calibration standard
can be calculated as in Eq 5. The concentrations of the both 12.4 LC-MS-grade methanol will serve as a solvent blank
primary and intermediate stock solutions in µg/g calculated in f
...