ASTM E3324-22

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: E3324 − 22
Standard Test Method for
Lipid Quantitation in Liposomal Formulations Using Ultra-
High-Performance Liquid Chromatography (UHPLC) with
Triple Quadrupole Mass Spectrometry (TQMS)
This standard is issued under the fixed designation E3324; 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 1.7 All observed and calculated values shall conform to the
guidelines for significant digits and rounding as established in
1.1 This test method describes the determination of lipid
Practice D6026.
components in liposomal formulations, which includes sample
1.8 Units—The values stated in SI units are to be regarded
solubilization in methanol followed by separation of the
asthestandard.Whereappropriate,c.g.sunitsinadditiontoSI
analytes using ultra-high-performance liquid chromatography
units are included in this standard.
(UHPLC) and detection with tandem mass-spectrometry (MS/
MS). This test method adheres to multiple reaction monitoring
1.9 This standard does not purport to address all of the
(MRM) mass spectrometry on a triple quadrupole mass spec-
safety concerns, if any, associated with its use. It is the
trometer (TQMS).
responsibility of the user of this standard to establish appro-
priate safety, health, and environmental practices and deter-
1.2 This test method is specific for liposomal formulations
mine the applicability of regulatory limitations prior to use.
containing cholesterol, 1,2- distearoyl-sn-glycero-3-
1.10 This international standard was developed in accor-
phosphoethanolamine-N-[methoxy (polyethylene glycol)-
dance with internationally recognized principles on standard-
2000] (DSPE- PEG 2000), and hydrogenated (soy) L-α-
ization established in the Decision on Principles for the
phosphatidylcholine (HSPC).
Development of International Standards, Guides and Recom-
1.3 This test method is applicable to report the absolute
mendations issued by the World Trade Organization Technical
concentrationsofcholesterol,DSPE-PEG2000,andHSPCand
Barriers to Trade (TBT) Committee.
their ratio (DSPE-PEG 2000: HSPC: cholesterol) in liposomal
2. Referenced Documents
formulations. Assessment of the stability of the analytes in
termsoftheirdegradationasaresultofoxidationorhydrolysis
2.1 ASTM Standards:
is beyond the scope of this test method.
D1193Specification for Reagent Water
D6026Practice for Using Significant Digits and Data Re-
1.4 This test method includes calibration and
cords in Geotechnical Data
standardization, sample preparation, UHPLC-TQMS
D7439Test Method for Determination of Elements in Air-
instrumentation, potential interferences, method validation
borne Particulate Matter by Inductively Coupled Plasma-
with acceptance criteria, sample analysis, and data reporting.
–Mass Spectrometry
1.5 The detection limits for cholesterol, DSPE-PEG 2000,
E177Practice for Use of the Terms Precision and Bias in
and HSPC using this test method are 5.3, 0.5, and 0.5 ng/g,
ASTM Test Methods
respectively.Inaddition,thequantitationlimitsforcholesterol,
E456Terminology Relating to Quality and Statistics
DSPE-PEG 2000, and HSPC are 10.6, 0.8, and 0.5 ng/g,
E682Practice for Liquid Chromatography Terms and Rela-
respectively.
tionships
E2490Guide for Measurement of Particle Size Distribution
1.6 This test method is intended for concentration ranges of
of Nanomaterials in Suspension by Photon Correlation
8-1600 ng/g for cholesterol, and of 2-400 ng/g for DSPE-PEG
Spectroscopy (PCS)
2000 and HSPC.
E3025Guide for TieredApproach to Detection and Charac-
terization of Silver Nanomaterials in Textiles
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 service@astm.org. For Annual Book of ASTM
Current edition approved Jan. 15, 2022. Published April 2022. DOI: 10.1520/ Standards volume information, refer to the standard’s Document Summary page on
E3324-22. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3324 − 22
2.2 Federal Standard: evaporate in the ion source of the mass spectrometer releasing
21 CFR 211.194(a)(2)Code of Federal RegulationsTitle 21, ions to the gas phase for analysis in the mass spectrometer (2).
Food and Drug administration, Department of Health and
3.1.11 fragment ion, n—product ion that results from the
Human Services, Drugs: Current Good Manufacturing
collision-induced dissociation of a preselected precursor ion
Practice for Finished Pharmaceuticals, Laboratory Re-
(3).
cords
3.1.12 internal standard, ISTD, n—chemical substance that
is added in a known amount to calibration standards and
3. Terminology
samples with unknown concentrations to correct for analyte
3.1 Definitions:
loss during sample preparation, ion suppression effects, source
3.1.1 accuracy, n—closeness of agreement between a test
fouling, or matrix effects during analysis (2).
result and an accepted reference value.
3.1.13 intermediate precision, n—closeness of agreement
3.1.1.1 Discussion—The term accuracy, when applied to a
between test results obtained under specified intermediate
set of results, involves a combination of random component
precision conditions. E177
and a common systematic error or bias component. E177
3.1.14 intermediate precision conditions, n—conditions un-
3.1.2 analyte, n—chemical constituent of interest in an
der which test results are obtained with the same test method
analytical procedure. E3025
using test units or test samples taken at random from a single
3.1.3 analytical instrument qualification, n—collection of
quantity of material that is as nearly homogeneous as possible
documented evidence that an instrument performs suitably for
and with changing conditions such as operator, measuring
its intended purpose (1).
equipment, location within the laboratory, and time.
3.1.4 baseline noise, n—combinationofhigh-frequencysig-
3.1.15 ionization effıciency, n—ratio of the number of ions
nal fluctuations and low-frequency signal drift that affects
generated to the number of molecules introduced into the ion
baseline stability.
source of a mass spectrometer (3).
3.1.4.1 Discussion—These signal fluctuations can originate
3.1.16 ion suppression, n—matrix effect in liquid
from line-voltage fluctuations, shot noise (Poisson noise) from
chromatography-massspectrometrythatresultsinadiminished
electronic circuits, solvent impurities, temperature instability,
analytical signal independent of the sensitivity or selectivity of
and other nonequilibrium effects. Noise is representative of
the mass analyzer (2).
detector response that is not related to responses from analytes
or matrix interferences. 3.1.17 limit of detection, LOD, n—lowestamountofanalyte
inasamplethatcanbedetectedbutnotnecessarilyquantitated
3.1.5 calibration curve, n—relationship between measured
under the stated experimental conditions.
response values and analytical concentrations of a standard or
3.1.17.1 Discussion—The limit of detection is usually ex-
reference material. D7439
pressed as the concentration of the analyte in the sample (4).
3.1.5.1 Discussion—A set of calibration standards are used
Different approaches to determine detection limits are possible
to construct a calibration curve, and the concentration of
that are based on the visual evaluation, signal-to-noise ratio, or
analyte present in an unknown sample can be determined by
standard deviation of the response and the slope. In the case of
comparing the detector response with the calibration curve.
instrumental analytical procedures, a common approach is to
3.1.6 calibration standards, n—set of solutions with known
compare the measured signals from samples having a known
analyte concentrations used to construct calibration curves.
low concentration of analyte with that of blank samples. The
3.1.7 carryover effect, n—systematic error that is derived
minimum concentration at which the analyte can be reliably
from the preceding sample injection introduced into the next
detected is established. Typically, acceptable signal-to-noise
sample affecting accurate quantitation.
ratio is 3:1 (4, 5).
3.1.8 cholesterol, n—steroidal organic compound that stabi-
3.1.18 linearity, n—ability of the analytical method (within
lizes the lipid bilayer in liposomal formulations.
a certain range) to obtain test results that are directly propor-
3.1.9 chromatogram, n—graphical presentation of detector tional to the concentration (amount) of the analyte in the
sample (3).
response plotted as a function of elution time or effluent
volume as the sample components elute from the column and
3.1.19 lipids, n—diverse group of organic compounds that
reach the detector.
are soluble in organic solvents but are insoluble in water.
3.1.10 electrospray ionization, ESI, n—sensitive ionization 3.1.19.1 Discussion—In this test method, lipids refer to
techniqueinwhichasolventsprayisformedbytheapplication cholesterol, DSPE-PEG 2000, and HSPC. The chemical struc-
of a high-voltage potential held between a stainless-steel tures of these three lipids are presented in Appendix X1.
capillary containing a solution and the instrument orifice and
3.1.20 liposomal formulation, n—product designed to assist
the axial flow of a nebulizing gas (typically nitrogen).
in the delivery of an active pharmaceutical ingredient either
3.1.10.1 Discussion—Solvent droplets from the spray
encapsulated or intercalated in the liposome.
3.1.20.1 Discussion—Formulated products can contain
vesicles having a single lipid bilayer (unilamellar), multiple
Available from www.govinfo.gov.
concentric lipid bilayers (multilamellar) or a mixture of unila-
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
this standard. mellar and multilamellar vesicles.
E3324 − 22
3.1.21 liposome, n—synthetic vesicle composed of one or quantity measured by mass spectrometry is not the ion’s mass
–1
more bilayers formed by amphipathic molecules such as divided by its electric charge (SI units kg C ).
phospholipids that enclose a central aqueous compartment.
3.1.31 peak area, n—area under a peak obtained from
Adapted from (6).
integration of a detector signal above the baseline for a given
3.1.22 lower limit of quantitation, LOQ, n—least amount of
component.
analyte in a sample which can be quantitatively determined
3.1.32 peak resolution (R ), n—measureofchromatographic
s
with suitable precision and accuracy.
separation of two components in a mixture. It is defined as the
3.1.22.1 Discussion—The LOQ is usually expressed as the
differencebetweentheretentiontimesofthetwopeaksdivided
concentration of analyte in the sample (4). This is also known
by their average peak width.
as lower limit of quantitation. Different approaches to deter-
t 2 t
~ !
R2 R1
mine detection limits are possible, which are based on the
R 5 2 3
S
~w 1 w !
visualevaluation,signal-to-noiseratio,orstandarddeviationof 1 2
the response and the slope. In the case of instrumental
where:
analytical procedures, a common approach is to compare the
t and t = retention time of the two components 1 and 2
R1 R2
measured signals from samples having known low concentra-
(t > t ), and
R2 R1
tionsofanalytewiththesignalsfromblanksamples.Usingthis
w and w = corresponding widths at the bases of the peaks
1 2
approach, the minimum concentration at which the analyte can
obtainedbyextrapolatingtherelativelystraight
be reliably quantified is established. A typically acceptable
sides of the peaks to the baseline (2).
signal-to-noise ratio is 10:1 (4, 5).
3.1.33 precision, n—closeness of agreement between inde-
3.1.23 mass spectrum, n—plot of the relative abundances of
pendent test results obtained under stipulated conditions. E177
ions forming a beam or other collection as a function of their
3.1.34 precursor ion, n—ion that reacts to form particular
m/z values (2).
product ions or undergoes specified neutral losses.
3.1.24 mass transition, n—specific pair of m/z values asso-
3.1.34.1 Discussion—The reaction can be of different types,
ciated with precursor and product ions.
including unimolecular dissociation, ion/molecule reaction,
3.1.25 matrix blank, n—substance that closely matches the
andchangeinchargestate,possiblyprecededbyisomerization
samples being analyzed with respect to matrix components but
(3).
has none of the analyte(s) of interest.
3.1.35 quadrupole, n—device used to separate ions accord-
3.1.25.1 Discussion—A matrix blank does not contain the
ing to their m/z using two pairs of parallel, equidistant poles
analyte(s) of interest but is subjected to all sample-processing
(rods) biased at equal, but opposite, electric potentials.
operations.Amatrix blank is used to determine the selectivity
of a test method and determine the absence of significant
3.1.36 qualifier transition, n—serves to confirm the identity
interference caused by matrix components.
of the analyte thereby enhancing the selectivity of the mea-
surement (2).
3.1.26 matrix effect, n—influence of one or more compo-
nents from the sample matrix on the measurement of the
3.1.37 quality control sample, QC, n—sample of known
analyte concentration or mass.
concentration that falls within the range of the calibration
3.1.26.1 Discussion—Matrix effects may be observed as
curve; it is prepared similar to calibration standards and
enhanced or suppressed detector responses compared with
injected at frequent intervals to check the performance of the
those produced by simple solvent solutions of the analyte (7,
instrument throughout the batch (see 3.2.1).
8).
3.1.38 quantifier transition, n—typically the most selective
3.1.27 method validation, n—process used to confirm that
transition; it is used for quantitation of the analyte (2).
ananalyticalprocedureusedforaspecifictestissuitableforits
3.1.39 recovery, n—extraction efficiency of an analytical
intended purpose.
process reported as a percentage of the known amount of an
3.1.28 mobile phase, n—liquid used to elute sample com-
analyte carried through the sample extraction and processing
ponents through the column that may consist of a single
steps of the method (5).
component or a mixture of components.
3.1.40 repeatability, n—precision of test results from tests
3.1.28.1 Discussion—The term eluent is often used for the
conductedwithintheshortestpracticaltimeperiodonidentical
preferred mobile phase. E682
materialbythesametestmethodinasinglelaboratorywithall
3.1.29 multiple reaction monitoring, MRM, n—application
known sources of variability conditions controlled at the same
of selected reaction monitoring to multiple product ions from
levels. Adapted from E177
one or more precursor ions (3).
3.1.41 reproducibility, n—precisionoftestresultsfromtests
3.1.30 m/z values, n—dimensionless quantity representing
conducted on identical material by the same test method in
the ratio of the mass of an ion (m) in atomic mass units (amu)
different laboratories. Adapted from E456
to its formal charge (z).
3.1.30.1 Discussion—The term “mass-to-charge ratio” has 3.1.42 response factor, n—measure of the relative mass
occasionally been used for m/z in the literature to represent the spectral response of an analyte compared to its internal
horizontal axis in a plot of a mass spectrum; however, the standard.
E3324 − 22
3.1.43 robustness, n—measure of change in the outcome of 3.2.1 batch, n—seriesofuninterruptedmeasurementswithin
an analytical procedure with deliberate and systematic varia- which the trueness and precision of the measuring system are
tions in any or all of the key method parameters that influence expected to be stable.
it. Adapted from E2490
3.2.2 test sample, n—final form of the test unit that is used
for single or multiple observations.
3.1.44 selected reaction monitoring, SRM, n—data acquired
3.2.2.1 Discussion—In this test method, a sample solubi-
from one or more specific product ions corresponding to m/z
lized with methanol, spiked with a known concentration of
selected precursor ions recorded via two or more stages of
ISTD, and diluted within the bracketed range is referred to as
mass spectrometry (3).
a test sample.
3.1.45 solvent blank, n—solution containing all reagents
3.2.3 test unit, n—portionofamaterialthatisobtainedfrom
used in sample dissolution in the same quantities used for
a primary material following a sampling procedure to acquire
preparation of blank and sample solutions.
test result(s) for the property(ies) to be measured.
3.1.45.1 Discussion—The solvent blank is used to assess
3.2.3.1 Discussion—In this test method, the original lipo-
contamination from the laboratory environment and character-
somal formulation to be tested for lipid quantitation is defined
ize spectral background from the reagents used in the sample
as a test unit.
preparation. D7439
3.3 Acronyms:
3.1.46 specificity, n—ability to assess unequivocally the
3.3.1 CoA, n—certificate of analysis
analyte in the presence of components that may be expected to
3.3.2 CRM, n—certified reference material
be present.
3.3.3 DMPC, n—1,2-Dimyristoyl-sn-glycero-3-
3.1.46.1 Discussion—Typically, such components might in-
phosphocholine
clude impurities, degradants, matrix material, and so forth (5).
3.3.4 DSPE-PEG 2000, n—1,2-Distearoyl-sn-glycero-3-
3.1.47 system suitability, n—determination of instrument
phosphoethanolamine-N-[methoxy (polyethylene glycol)-
performance in a particular procedure (for example, sensitivity
2000]
and chromatographic retention) by analyzing a set of appro-
priate reference standards before the analytical run. 3.3.5 DSPE-PEG 550, n—1,2-Distearoyl-sn-glycero-3-
phosphoethanolamine-N-[methoxy (polyethylene glycol)-550]
3.1.48 tandem mass spectrometry, MS/MS, n—acquisition
3.3.6 ESI, n—electrospray ionization
and study of the spectra of the product ions or precursor ions
ofm/zselectedionsorprecursorionsofaselectedneutralmass
3.3.7 HSPC, n—hydrogenated (soy) L-α-
loss (3).
phosphatidylcholine
3.1.48.1 Discussion—MS/MS can be accomplished using
3.3.8 IS, n—intermediate stock
instruments incorporating more than one mass analyzer (tan-
3.3.9 ISTD, n—internal standard
dem mass spectrometry in space) or in trap instruments
3.3.10 LC-MS, n—liquid chromatography-mass spectrom-
(tandem mass spectrometry in time). In this test method, a
etry
TQMS has been used, which contains two quadrupole mass
analyzers in a series, separated by a collision cell. Precursor 3.3.11 LOD, n—limit of detection
ions are selected by the first quadrupole mass analyzer. The
3.3.12 LOQ, n—limit of quantitation
selectedprecursorion(s)isthenfragmentedinthecollisioncell
3.3.13 MRM, n—multiple reaction monitoring
in the presence of inert gas, such as nitrogen or argon by a
3.3.14 MS/MS, n—tandem mass spectrometry
process known as collision-induced dissociation. Specific
productionsareselectedbythefinalquadrupolemassanalyzer
3.3.15 m/z, n—mass-to-charge ratio
and passed to the detector. Thus, only analyte ions having a
3.3.16 OSHA, n—Occupational Safety and HealthAdminis-
specifiedmasstransition(precursor/productionpair)canreach
tration
thedetector,resultinginthehighspecificityofTQMSmethods
3.3.17 PS, n—primary stock standard
(2).
3.3.18 QC, n—quality control
3.1.49 total ion current chromatogram, TIC,
3.3.19 RCF, n—relative centrifugal force
n—chromatogram created by plotting the total ion current in a
series of mass spectra recorded as a function of retention time.
3.3.20 RSD, n—relative standard deviation
3.1.49.1 Discussion—Total ion current is the sum of all the
3.3.21 SD, n—standard deviation
separateioncurrentscarriedbytheionsofdifferentm/zvalues
3.3.22 SDSs, n—safety data sheets
contributing to a complete mass spectrum (3).
3.3.23 S/N, n—signal-to-noise ratio
3.1.50 triple quadrupole mass spectrometry, TQMS,
3.3.24 SRM, n—selective reaction monitoring
n—tandem mass spectrometer comprising two transmission
3.3.25 TIC, n—total ion chromatogram
quadrupole mass spectrometers in series with a non-selecting
transmission quadrupole (or other multipole) between them to
3.3.26 TQMS, n—triple quadrupole mass spectrometry
act as a collision cell (3).
3.3.27 UHPLC, n—ultra-high-performance liquid chroma-
3.2 Definitions of Terms Specific to This Standard: tography
E3324 − 22
3.3.28 ULOQ, n—upper limit of quantitation production, thereby facilitating safe and efficient drug devel-
opment and regulatory review.
3.3.29 WISM, n—working internal standard mixture
5.3 UHPLC-MS/MS measurements are analytically more
3.3.30 WSM, n—working standard mixture
sensitive and specific for lipid analysis compared to other
contemporary techniques using universal detectors, such as a
4. Summary of Test Method
charged aerosol or an evaporative light-scattering detector. For
4.1 Cholesterol,DSPE-PEG2000,andHSPCinaliposomal
liposomes, MS/MS has further advantages over ultraviolet
formulation are solubilized in methanol at 1:100 dilution by
detectors,aslipidslackchromophoresfordetection.Inthistest
volume followed by vortex mixing. The solubilized sample is
method, TQMS has been used as the MS/MS technique of
further diluted to appropriate concentrations to fit the calibra-
choice because of its high selectivity, sensitivity, S/N,
tion range and amended with ISTDs. The diluted sample is
accuracy, and broad linear range of quantitation, thereby
subjected to quantitative analysis using UHPLC-TQMS. Be-
allowingreproduciblequantitationoftheanalytes,especiallyat
fore sample analysis, the test method is validated for linearity,
low concentrations.
precision, accuracy, specificity, LOD, and LOQ.
5.4 According to the Current Good Manufacturing Practice
4.2 Calibration curves are established for DSPE-PEG 2000
regulations [21 CFR 211.194(a)(2)], users are required to
and HSPC in the range of 2–400 ng/g. Because of the low
verify the suitability of the test method under actual conditions
ionization efficiency, the range of the calibration curve for
of use. Validation should assess the suitability of the test
cholesterol(8–1600ng/g)wasexpandedtobeaboutfourtimes
method for the product matrix, recovery of the analytes from
greater than the calibration range for DSPE-PEG 2000 and
the product matrix, suitability of chromatographic conditions
HSPC. The analytes are separated on a bridged ethylsiloxane/
and column, appropriateness of the detector signal response,
silica hybrid (BEH) C18 column using UHPLC, ionized using
specificity, limit of detection and quantitation, accuracy, and
an ESI positive source and quantified using MRM mode on a
precision. The user may need to optimize method parameters
TQMS.
and cross validate if a different chromatography column,
ionization method, or mass analyzer is used.
4.3 The target analytes and ISTDs are identified by the
retention time and two MRM transitions. HSPC and DMPC
6. Interferences
use only one MRM transition because of less-sensitive or
nonexistent secondary transition. The analytes are quantitated
6.1 Method interferences may be introduced by impurities
in calibration standards and sample using the primary MRM
present in solvents, reagents, glassware, and other apparatus
transition with ISTD calibration. The final report issued for
used during sample preparation and instrumental analysis.
each sample lists the absolute concentration (ng/g) of the
These impurities may result in elevated baseline noise or
individuallipidsandtheirmassratio(DSPE-PEG2000:HSPC:
interfering peaks. The presence and magnitude of such inter-
cholesterol).
ferences are determined by routine analysis of solvent and
laboratory blanks under the same conditions as the samples.
5. Significance and Use
6.2 Matrix interferences may be caused by sample compo-
5.1 Liposomes are vesicles of nanoscale dimensions, com-
nents and contaminants from sampling devices and storage
posed of lipid bilayers, which are used for various diagnostic
containers. The extent of matrix interferences may vary con-
and therapeutic applications (9). The growing interest in
siderably depending on the source and application of the
liposomal formulations in the delivery of various drugs,
liposomal formulations. The analysis of matrix spikes is
antisense oligonucleotides, cloned genes, or recombinant pro-
critical for determining the impact of matrix interferences. If
teins by the biopharmaceutical industry, warrants QC and
the user observes any matrix effects under the recommended
thorough characterization of the constituent lipids. Lipid
test conditions, it may be necessary to modify the sample
structure, composition, and concentration are key attributes in
preparation procedure to remove the interfering compounds
determining the quality and efficacy of a liposomal drug
from the sample or to optimize the chromatographic param-
product as they influence the stability of liposomes, drug
eters to avoid the co-elution of target analytes and interfering
loading, release kinetics, biodistribution, and pharmacokinetic
excipients. The Bligh-Dyer method, as described in Appendix
properties (9). Cholesterol modulates the lipid membrane
X2, could be adopted to remove water-soluble excipients in
fluidity, elasticity, and permeability; hence, it plays a key role
liposomal formulations, if necessary (12).
in controlled drug release and increased stability of the
6.3 This test method requires the use of high-quality con-
liposome (10).
sumables and LC-MS grade reagents and solvents.
5.2 This test method provides a rapid and reliable protocol
6.4 Itisnecessarytouseahigh-qualitynitrogensource(>95
for the determination of cholesterol, DSPE-PEG 2000, and
% purity) that is free from water vapor, particles, and nonvola-
HSPC in liposomal formulations using UHPLC-TQMS. As-
tile hydrocarbons, such as compressor oils. For the collision
sessment of the stability of the analytes in terms of their
cell, a high purity (>99 %) gas source, in this case argon, is
degradation profiles is not included in this test method (11).
required.
This test method will benefit the biopharmaceutical industry in
ascertaining quality assessment of liposomal formulations and 6.5 To minimize baseline noise and ion-suppression effects,
monitoring batch-to-batch consistency for large-scale it is recommended to clean the autosampler injection port after
E3324 − 22
each injection for 10 s, wash the column frequently with conform to the specifications of the Committee on Analytical
appropriate solvents, and keep the ESI source clean. The Reagents of the American Chemical Society where such
auto-sampler injection port, column, solvent pumps, and the specifications are available. Other grades may be used, pro-
MS should be maintained as per the manufacturer’s instruc- vided it is first ascertained that the reagent is of sufficiently
tions. high purity to permit its use without lessening the accuracy of
the determination.
6.6 It is recommended to clean solvent reservoir bottles
routinely and use freshly prepared mobile phase solutions to 8.2 Ammonium formate deliquescent crystals, LC-MS
avoid any contamination.
grade.
6.7 Chemicals with high purity shall be used for the 8.3 Acetonitrile, LC-MS grade.
preparation of lipid calibration standards. When feasible, it is
8.4 Formic acid, LC-MS grade.
recommended that higher-order reference standards (for
8.5 Methanol, LC-MS grade.
example, CRMs) be acquired for calibration standards. If
referencematerialsarenotavailable,high-qualitycrystallineor
8.6 Water, LC-MS grade.
lyophilized chemicals of known purity can be used. For
8.6.1 Purity of Water—Unless otherwise indicated, refer-
isotope-labelled ISTDs, the use of a stable analyte with high
ences to water shall be understood to mean reagent water
isotopic purity is recommended. The presence of unlabeled
conforming to Type I of Specification D1193. It shall be
analyte should be checked, and if detected, the potential
confirmed that this water does not contain contaminants at
influence should be evaluated during method verification (13).
concentrations that would interfere with the analysis. LC-MS-
grade water prefiltered with a ≤0.2 µm filter was used to
7. Apparatus
develop this test method.
7.1 LC/MS/MS System:
NOTE1—Pre-mixedLC-MS-gradesolventswith0.1%formicacidmay
7.1.1 LC System—A UHPLC system is required to analyze
be used, if commercially available. For example, “methanol with 0.1 %
samples. It should include a sample injection system, a solvent
formic acid.”
pumping system capable of mixing solvents, an in-line degas-
8.7 Gases—Ultrapure nitrogen and argon.
ser module, a sample compartment capable of maintaining the
8.8 Lipid Standards:
required temperature, and a temperature-controlled column
compartment.AnyUHPLCsystemthatcanperformattheflow 8.8.1 Cholesterol, ≥99 % pure, powder form.
8.8.2 DSPE-PEG 2000, ≥99 % pure, powder form.
rates, pressures, controlled temperatures, sample volumes, and
other requirements of the test method shall be used. 8.8.3 HSPC, ≥99%pure,consistingofC16:0(HSPC1)and
C18:0 (HSPC 2) fatty acids, powder form.
7.1.2 Analytical Column—A reversed phase C-18 column,
13 nm pore size, 1.7 µm particle size, 2.1 mm × 50 mm
8.9 ISTDs:
(internal diameter x length) or equivalent column that can
8.9.1 Cholesterol-d7, powder form.
resolve all analytes of interest with R ≥ 1.5 is acceptable. The
s
8.9.2 DMPC, powder form.
retention times and order of elution may change depending on
8.9.3 DSPE- PEG 550, powder form.
the column being used.
9. Hazards
7.1.3 Guard Column—A C-18 pre-column, preferably with
the same packing as the analytical column, shall be installed
9.1 Standard protective measures are required while han-
between the injector and the analytical column.
dling liposomal formulations and organic solvents. This test
7.1.4 MS/MS System—An MS/MS system capable of MRM
methodusesflammableorganicsolvents,suchasmethanoland
analysis and performing at the requirements specified in this
acetonitrile. Precautions shall be taken to avoid direct contact
test method shall be used (see 11.5).
with skin and inhalation of solvents. The user is advised to
conduct a hazard assessment to determine appropriate safety
7.2 Analytical balance that can accurately weigh with
protocolsandpersonalprotectiveequipment(PPE),andfollow
≤0.0001 g readability.
applicable regulations (for example, OSHA) , supplier SDSs,
7.3 Vortex mixer.
institutional requirements, and recommended procedures per-
7.4 Mechanical pipettes and disposable pipette tips ranging
taining to safe handling and disposal of all chemicals used in
from 0.002 mL to 10 mL.
this test method.
7.5 Ultrasonic water bath.
10. Preparation of Mobile Phases
7.6 Solvent reservoir bottles, 1 L.
10.1 Mobile Phase A — 60 % Acetonitrile: 40 % Water
7.7 Polypropylene tubes, 15 mL.
Containing 0.1 % Formic Acid and 10 mmol/L Ammonium
7.8 Amber glass vials, 10 mL and 20 mL. Formate:
7.9 Amber autosampler vials (2 mL) with polytetrafluoro-
ACS Reagent Chemicals, Specifications and Procedures for Reagents and
ethylene silicone septa-fitted caps.
Standard-Grade Reference Materials, American Chemical Society, Washington,
DC. For suggestions on the testing of reagents not listed by theAmerican Chemical
8. Reagents and Materials
Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset,
8.1 Reagent grade chemicals shall be used in all tests.
U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharma-
Unless otherwise indicated, it is intended that all reagents copeial Convention, Inc. (USPC), Rockville, MD.
E3324 − 22
10.1.1 Rinse an empty and clean solvent reservoir bottle (1 tainedinapowered-onstateunlessrecommendedotherwiseby
L) thoroughly with LC-MS-grade water and dry. Label the the manufacturer. The TQMS is typically maintained in
bottle to include the mobile phase composition and date of standby mode.
preparation.
11.2 LC Chromatograph Operating Conditions—The injec-
10.1.2 To prepare 1 L of mobile phase A, weigh 0.630 6
tion volume of all calibration standards and samples is 5 µL.
0.006 g of LC-MS-grade ammonium formate and transfer it to
The flow rate of the mobile phase should be set at 0.4 mL/min
the rinsed and dried solvent reservoir bottle.
and the column temperature adjusted to 60.0°C. The LC
10.1.3 Measure 400 mL of LC-MS-grade water using a
gradient conditions are shown in Table 1.
measuring cylinder and transfer it to the solvent reservoir
11.3 LC Sample Manager Conditions:
bottle.
11.3.1 Needle Wash—Prime the sample syringe and needle
10.1.4 Measure 600 mL of LC-MS-grade acetonitrile using
with LC-MS-grade acetonitrile/water/methanol, 1:1:1 (v/v/v),
a measuring cylinder. Transfer to the solvent reservoir bottle
toremovecontaminantsfrominsideandoutsidetheneedleand
and mix thoroughly.
the injection port. This also helps purge air from the lines.
10.1.5 Add 1 mLof LC-MS-grade formic acid to the bottle
11.3.2 Seal Wash—90 % water, 10 % acetonitrile; time: 2
and mix thoroughly. Skip this step if using 0.1 % formic acid
min.
premixed LC-MS-grade water and acetonitrile (see Note 1).
11.3.3 Autosampler Purge—60 % acetonitrile and 40 %
10.1.6 Degas mobile phaseAfor 10 min using an ultrasonic
water containing 0.1 % formic acid.
bath at atmospheric pressure.
11.3.4 Temperature—Column, 60ºC; Sample compartment,
12ºC.
NOTE 2—The use of an in-line degasser module in the UHPLC stack
11.3.5 Wash and purge specifications from specific instru-
helps to achieve a stable baseline during the analytical run. Sparging with
helium can also be used as an alternative to the ultrasonic and in-line
ment manufacturers should be followed to eliminate sample
vacuum degassing combination as used in this test method.
carryover in the analysis of lipids.
10.2 Mobile Phase B — Methanol Containing 0.1 % Formic
11.4 Column Conditioning—Once the desired column tem-
Acid and 10 mmol/L Ammonium Formate:
perature (60ºC) is achieved, equilibrate the column using a
10.2.1 Rinse an empty and clean solvent reservoir bottle (1
40:60 mixture of mobile phases A:B at a flow rate of 0.4
L) thoroughly with LC-MS-grade methanol and dry. Label
mL/min for at least 30 min before starting the batch with the
appropriately with necessary details (see 10.1.1).
elution set to waste.
10.2.2 To prepare 1 L of mobile phase B, weigh 0.630 6
11.5 MS Parameters:
0.006 g of LC-MS-grade ammonium formate and transfer it to
11.5.1 To acquire the maximum number of data points per
the rinsed and dried solvent reservoir bottle.
MRM channel with optimum sensitivity, the tunable param-
10.2.3 Measure 1 Lof LC-MS-grade methanol in a measur-
eters shall be optimized for the instrument being used for the
ing cylinder. Transfer to the solvent reservoir bottle and mix
analysis. Each peak requires at least ten scans per peak for
thoroughly.
adequate quantitation. This test method contains three target
10.2.4 Add 1 mLof LC-MS-grade formic acid to the bottle
analytes and three ISTDs for respective lipids, which are split
and mix thoroughly (see 10.1.5).
into different MRM experiment windows to optimize the
10.2.5 DegasmobilephaseBfor10minusinganultrasonic
number of scans and sensitivity. MS tune parameters used in
bath at atmospheric pressure.
thedevelopmentofthistestmethodareprovidedinTable2for
information only. These conditions shall be optimized by the
10.3 Useaninlet-linefrit(0.2µmporesize)foreachmobile
user. The instrument is set in the electrospray positive source
phase and keep the inlet at the bottom of the reservoir to
setting. Variable parameters including retention times, MRM
remove large particles.
transitions, cone voltage, and collision energy used to develop
10.4 Prepare fresh mobile phase solutions before UHPLC-
this test method are shown in Table 3. As HSPC is composed
TQMS analysis.
of two compounds HSPC-1 and HSPC-2 at unknown ratios in
10.5 Prime the solvent lines and the pump system with the calibration standards and samples, it was quantified by
freshly prepared Mobile PhasesAand B for 2 min to eliminate
air bubbles and ensure a steady flow out of the vent tube.Any
TABLE 1 Gradient Conditions for LC
glassware used to prepare or store a mobile phase shall be
Mobile Mobile
Time Flow Rate
rinsed with respective LC-MS grade solvent before use (14),
Phase A Phase B
(min) (mL/min)
forexample,waterformobilephaseAandmethanolformobile (%) (%)
phase B. 0.0 0.4 40 60 Diverted to waste
1.0 0.4 20 80
2.0 0.4 15 85 Directed to MS
11. Preparation of Apparatus
5.2 0.4 15 85
5.3 0.4 10 90
11.1 Analytical Instrument Qualification—The UHPLC-
6.2 0.4 5 95
TQMS system shall be maintained periodically following the 6.5 0.4 0 100
8.5 0.4 0 100 Diverted to waste
manufacturer’s recommendation to obtain reliable quantitative
8.8 0.4 40 60
data. Typically, the vacuum systems, high-voltage power
10.0 0.4 40 60
supplies, computers, and data collection systems are main-
E3324 − 22
TABLE 2 Typical MS Tune Parameters NOTE 5—The user is required to provide a CoA or an equivalent
alternative information on the source, purity, storage conditions, retest/
Electrospray Positive Mode
expiration date, and lot number of the reference standard or high-purity
Capillary voltage 2.85 kV
chemical to ensure quality and stability (13). A full scan MS of standard
Cone Variable depending on analyte (Table 3)
solutions could be used to check for decomposition or conversion
Source temperature 150°C
products. When using expired reference standards or a high-purity
Desolvation temperature 500°C
Desolvation gas flow 1000 L/h chemical, the user should provide an updated CoA or reestablish the
Cone gas flow 150 L/h
identity, purity, and stability of the chemicals. For ISTDs, the user does
Low mass resolution 1 2.52
not have to provide a CoA or evidence of purity if the suitability for use
High mass Resolution 1 14.96
isdemonstrated,forexample,alackofinterferencewithananalyte.Stock
Ion energy 1 0.0
and working solutions can only be prepared from reference standards that
Entrance energy 30 V
arewithinthestabilityperiodasdocumentedintheCoA,eitherexpiration
Collision energy Variable depending on analyte (Table 3)
date or retest date.
Exit energy 30 V
Low mass resolution 2 2.86
NOTE 6—In the working standard mixtures (WSMs) and calibration
High mass resolution 2 14.86
standards, concentrations of cholesterol should be four times higher than
Ion energy 2 0.9
the concentrations of DSPE-PEG 2000 and HSPC, because of its low
Multiplier 504.76
ionization efficiency with the ESI. In the working ISTD mixtures
Inter-scan delay 0.02 s
(WISMs) and calibration standards, cholesterol-d7 should also be four
Dwell time 0.007 s
times higher than DSPE-PEG 550 and DMPC.
12.1 Preparation of Calibration Standards—To prepare the
adding the response of the two individual compounds using
calibration curve, analyze eight calibration standards named
their respective MRM transitions. Levels (LV) 1–8 which contain cholesterol, DSPE-PEG2000,
11.5.2 TQMS Stabilization—Turn on the nitrogen and argon
and HSPC at nominal concentrations ranging from 8–1600
gas source and change the TQMS system from standby mode
ng/g for cholesterol and 2–400 ng/g for DSPE-PEG 2000 and
to operate mode. Open the optimized MS method file for this
HSPC as shown in Table 4 (rows 1–3). ISTDs are used to
test method. The TQMS system shall be allowed time to
account for experimental drift in the test method caused by
stabilizewithallpredefinedparameterssetforthistestmethod.
matrix interferences that may result in ion enhancement or
The user shall verify that all the parameters are within the set
suppression. Three ISTDs—cholesterol-d7, DSPE-PEG 550,
range.
and DMPC—are added to the analytical calibration standards
and samples at nominal concentrations of ≈ 100, 25, and 25
11.6 After stabilizing the TQMS and equilibrating the col-
ng/g, respectively, as shown in Table 4 (rows 4–6). The
umn to obtain the stable desired temperature and pressure
following steps were used to produce calibration standards
(pressure fluctuations should be <3 bar), direct the UHPLC
with the concentration levels (LV1-LV8) shown in Table 4.
flow to the TQMS ESI source.
11.7 System Suitability Check:
12.2 Preparation of Stock Solutions—Primary stock stan-
11.7.1 After conditioning and stabilizing the UHPLC-
dard (PS) solutionsarepreparedforeachanalytefromstandard
TQMSsystemandbeforebeginningeachbatchanalysis,inject
materials or purchased as certified solutions (see Note 5).
a QC sample three to five times through the column.
These solutions are then diluted to prepare individual interme-
11.7.2 Process the data and make sure the analyte/ISTD
diate stock standard (IS) solutions.TheISsolutionsareusedto
peak area ratios and retention times are within the expected
prepare WSMs containing cholesterol, DSPE-PEG 2000, and
ranges. The system suitability acceptance criteria for the test
HSPC, at three concentration levels and a WISM containing
method are set at a relative standard deviation (RSD) <6 % for
cholesterol-d7, DSPE-PEG 550, and DMPC.Aliquots of three
analyte/ISTDpeakarearatiosand 60.5minforretentiontime.
WSMs (A, B, and C) are mixed with the WISM solution and
The S/N for the detector signal response shall at least be 10:1.
diluted with solvent to prepare calibration standards (Table 5).
11.7.3 Once the UHPLC and MS systems pass the system
12.2.1 Powder stocks of the chemicals stored in the freezer
suitability check, the sample batch containing the set of
at –20ºC or per manufacturer’s recommendation should be
calibration standards and test samples shall be run on the
allowedtoequilibrateatambienttemperaturefor15minbefore
instrument.
weighing.
12. Calibration and Standardization 12.2.2 PS Solutions—Prepare 10 mL of individual PS solu-
NOTE 3—Conditioning the pipette tip with appropriate solvents before tions (≈1000 µg/g) of cholesterol, DSPE-PEG 2000, HSPC,
the transfer of calibration standards for weighing, working promptly with
cholesterol-d7, DSPE-PEG 550, and DMPC in 20 mL amber
the stock solutions, and weighing volatile liquids in securely capped
glass vials.
containers using a secondary container is recommended.
12.2.2.1 Rinse six empty 20 mL amber glass vials with
NOTE 4—All solutions in this test method are prepared gravimetrically
using an analytical balance (≤0.0001 g accuracy). Hence, the user is
deionized water and dry thoroughly.
responsible for recording weights carefully at every step of preparation of
12.2.2.2 Label each vial with the corresponding analyte i
calibration standards and test samples. Although the volumetric sample
(where i = cholesterol, DSPE-PEG 2000, HSPC, cholesterol-
preparation closely agrees with the gravimetric sample preparation, it is
known that a 1–5 % error can be introduced during small volume
d7, DSPE-PEG 550, or DMPC) and the estimated concentra-
transfers, especially for volatile or semi-volatile organic solvents which
tion of the analyte.
maybiasthequantitation.Theanalyticalbalanceprovidesbettermeasure-
12.2.2.3 Weigh each empty capped vial on an analytical
mentresolution(thatis,moresignificantfigures)thanmechanicalpipettes
and offers better accuracy (15). balance and record the mass to the nearest 0.0001 g as W .
0i
E3324 − 22
TABLE 3 Retention Times, MRM Ions, and Analyte-Specific MS Parameters
Retention Primary/ MRM Transition Cone, Collision,
Analyte
Time, min Confirmatory (Parent >Product) V eV
DMPC 2.56 Primary 678.60 > 184.02 38 30
Cholesterol-d7 2.68 Primary 376.41 > 147.08 38 24
Confirmatory 376.41 > 160.96 38 22
Confirmatory 376.41 > 95.24 38 30
Cholesterol 2.70 Primary 369.55 > 161.15 38 22
Confirmatory 369.55 > 95.10 38 26
Confirmatory 369.55 > 147.25 38 24
DSPE-PEG 2000 3.82 Primary 607.55 > 95.16 76 30
Confirmatory 607.55 > 109.02 76 32
Confirmatory 726.30 > 607.55 40 10
HSPC-1 4.62 Primary 762.61 > 184.14 32 30
DSPE-PEG 550 4.66 Primary 607.45 > 94.99 70 30
Confirmatory 607.45 > 71.11 70 32
Confirmatory 751.06 > 607.45 40 10
HSPC-2 5.80 Primary 790.59 > 184.08 32 30
TABLE 4 Concentrations of Calibration Standards, ng/g
Analyte/ISTD LV1 LV2 LV3 LV4 LV5 LV6 LV
...