Quantification of Phospholipid Degradation Products in Liposomal Pharmaceutical formulations by Ultra Performance Liquid Chromatography- Mass Spectrometry (UPLC-MS)
Dumindika A. Siriwardanea, Changguang Wanga, Wenlei Jiangb, Thilak Mudaligea
a, Arkansas Laboratory, Office of Regulatory Affairs, U.S. Food and Drug Administration,
Jefferson, Arkansas, 72079
b, Office of Research and Standards, Office of Generic Drugs, Center for Drug Evaluation and
Abstract
Identification and quantification of excipient related degradation products in the liposomal formulation is important, as they may impact the safety and efficacy of the drug. Phospholipids are one of the major excipients in liposome drugs composing the lipid bilayer, and they are vulnerable to oxidation and hydrolysis reactions. Since phospholipids with saturated fatty acid chain were preferred in most of liposome drug products, the major degradation pathway of phospholipids in liposome formulations are limited to hydrolysis of phospholipids into free fatty acids and lysophospholipids. These hydrolyzed degradation products may form during manufacturing and/or long-term storage of liposomal formulations. Herein, we report development and application of accurate and sensitive methods that can be utilized for the quantitation of saturated free fatty acids (FFA 18:0 and FFA 16:0), lysophosphocholines (LPC 18:0 and LPC 16:0), and lysophosphoglycerol (LPG 18:0) in liposomal formulations. The free fatty acids were separated using a C8 column whereas the LPCs and LPGs were separated using a C18 stationary phase upon direct injection without the need of lipid extraction process. Each analyte was quantified by Q-TOF mass spectrometry. This method was validated according to USP compendial procedures and has been applied to the analysis of four commercial liposomal pharmaceutical formulations. The limit of quantitation (LOQs) of FFA 16:0, FFA 18:0, LPC 16:0, LPC 18:0 and LPG 18:0 are 5 ng/mL, 5 ng/mL, 6.5 ng/mL, 7.0 ng/mL, 10 ng/mLrespectively. Compared to CAD (Charge Aerosol Detector) and ELSD (Evaporative Light Scattering Detector) detection methods in ppm levels, this ultra-performance liquid chromatography (UPLC)- Mass Spectroscopy (MS) method displays precise determination of lysophospholipids in the liposomal formulations with higher accuracy and sensitivity.
1. INTRODUCTION
Liposomes have been successfully used as drug delivery vehicles to improve therapeutic efficacy and reduce the toxicity of active pharmaceutical ingredients (APIs) (Hofheinz et al., 2005; Wang et al., 2012). For instance, reduction of toxicity of daunorubicin, and doxorubicin by encapsulation in liposomes has been well established (Forssen and Tokes, 1981; Hamilton et al., 2002; McMenemin et al., 2002). Liposomes have been investigated as potential carriers for the site-specific delivery of enzymes, macromolecules, antimicrobial compounds, anti-tumor agents, and biological response modifiers as well (Kraft et al., 2014). The diversity in the design of liposomes by composition, structure, and size make it possible to tailor a drug-delivery system which is more efficacious than use of the unencapsulated drug and has improved the half-life in the circulation system by evading immune system (Sercombe et al., 2015). Phospholipids serve as the major structural component of liposome bilayers. Phospholipids are amphiphilic molecules having hydrophobic tails and hydrophilic head groups. The glycerol acts as the backbone of phospholipids having two fatty acid groups attached to the first and second carbon as esters and a phosphate ester on the third carbon (Figure 1). During manufacturing and storage, phospholipids in the liposome drug formulation, particularly phosphatidylcholine (PC) and phosphatidylglycerol (PG) may potentially be hydrolyzed when exposed to excess moisture, heat, and light. For instances, PCs can be hydrolyzed to lysophosphatidylcholines (LPCs) and free fatty acids (FFAs), whereas LPGs and FFAs are formed through the hydrolysis of PGs (Scheme 01).
It is reported that LPCs can interfere with lipid bilayer dynamics and can eventually affect drug release rate and stability of the formulations. For instance, LPC can modulate the phase transition temperature and are used in thermosensitive liposomes to set an appropriate phase transition temperature for temperature-controlled release(Huang et al., 2005; Koklic and Trancar, 2012; Needham and Zhelev, 1995; Qiao et al., 2006; Zylberberg and Matosevic, 2016).
According to FDA’s Guidance for Industry: Liposome Drug Products : Chemistry, Manufacturing, and Controls; Human Pharmacokinetics and Bioavailability; and Labeling Documentation, section 5c (FDA, 2018), the impurity information of lipid components includinglysophospholipids and free fatty acids used to manufacture the drug product should be provided. In section 6d. (Drug Product Specification), “Degradation products related to the lipids (e.g., lysolipids) or drug substance,” the sound scientific appraisal of potential degradation products should be characterized including free drug substances, degradation products related to the drug substance and lipids, and residual solvents. . Therefore, a sensitive analytical method to quantify these trace amounts of lipid impurities in liposome formulations is needed to ensure liposome drug quality.
A variety of analytical techniques have been used for the detection of lysolipids in various sample matrixes such as soy phytochemical extract, mice serum, rat blood and serum (Fang et al., 2003; Jin et al., 2014; Yokoyama et al., 2000; Yu et al., 2010). Most of the methods are used in qualitative analysis of complex lysophospholipids in biological samples rather than quantitative analysis. In the early stage, MALDI-TOF, NMR and high-performance thin layer chromatography (HPTLC) (Freyburger et al., 1989; Schiller et al., 2001) were used for the qualitative analysis of lysophospholipids. (Delmonte et al., 2009). Gas chromatogram (GC) coupled with various detection technique such as flame ionization (Kail et al., 2012; Zhang et al., 2015) and electron ionization mass spectrometry (Rigano et al., 2016; Thurnhofer and Vetter, 2005) has been used to separate and quantitate free fatty acids. FFAs are nonvolatile compounds; therefore, derivatization to esters are essential. The use of reverse-phase high-performance liquid chromatography (HPLC) coupled with evaporative light-scattering detection (ELSD) has been applied for the separation and detection of lysophospholipids containing different acyl chains and head groups from complex mixtures (Hoischen et al., 1997), as well as quantitative analysis of LPC (Tam et al., 2018). However, these quantitative techniques still suffer from low sensitivity and poor selectivity for lysophospholipids as most of the detectors are molecule nonspecificuniversal detectors. The recent emergence of UPLC with small column particles has enabled much higher resolution and decreased analysis time relative to HPLC.
By contrast, electrospray ionization mass spectrometry (ESI–MS) stands out as a suitable quantitative analytical technique due to the easily ionizable nature of the polar head group of Lyso compounds and specificity associated with molecular mass (Liebisch et al., 2002). The soft ionization achieved using ESI–MS detects intact molecules with very high sensitivity and makes it an ideal technique for the analysis for phospholipids in biological samples.
Herein, our main objective was to develop a sensitive and reproducible method based on UPLC-QTOF to characterize phospholipids degradation products including FFAs, LPCs, and LPGs, as part of the evaluation of the liposomal drug formulations.
2. Experiments
2.1 Reagents and solvents
All solvents were LCMS grade (Optima®) and were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Hydrogenated soybean phosphatidylcholine (HSPC, Lot# 840058-03-037), Distearylphosphatidylglycerol (DSPG, Lot# 840465-01-114),Lysophosphatidylcholines (LPC) of 16:0 (Lot# 855675-01-081), LPC18:0 (Lot# 855775-01- 083), and LPC19:0 (Lot# 855776-01-022) and Lysophosphatidylglycerol (LPG) 18:0 (Lot# 858124P-500 mg-B-044), LPG16:0 (Lot# 858122P-500 mg-B-084), were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). HPLC grade chloroform was purchased from Alfa Aesar Inc. (Ward Hill, MA, USA). Ammonium formate (Lot# 172988), ammonium acetate (Lot# 126548), and formic acid (Lot# 142555) were LCMS grade (Optima®) andpurchased from Fisher Scientific (Fair Lawn, NJ, USA). Free fatty acid (FFA) 18:0 (Product# 85679 Lot# BCBW8354), FFA 16:0 (Product# 76119 Lot# BCBX4516), and FFA 16:0-d31(Product# 366897 Lot# MBBCW5485) were purchased from Sigma-Aldrich Inc (St. Louis, MO, USA). Liposomal pharmaceutical formulations were purchased from the pharmacy and they were delivered and stored under appropriate recommended conditions.
2.2 UPLC-MS conditions
Chromatographic analysis was performed using an Agilent 1290 Infinity™ UPLC system (Santa Clara, CA, USA) coupled to an Agilent 6550 QTOF Mass Spectrometer. Agilent Mass Hunter data acquisition, qualitative analysis, and quantitative analysis software were used for instrumental control, data control, data acquisition, qualitative and quantitative data analysis. The MS parameters were set as follows: sheath gas temperature: 250°C, sheath gas flow: 14 L/min, nebulizer: 60 psi, capillary voltage: 3500 V, and fragmentor: 175 V. LPCs were analyzed in positive mode, whereas LPG and FFAs were analyzed in negative mode.
For the determination of LPCs, ACQUITY UPLC® CSH™ C18 column (100 X 2.1 mm; particle size 1.8 µm, Waters, Milford, MA) was used. 10 mM ammonium formate in water with 0.1% formic acid (A) and 10 mM ammonium formate in methanol with 0.1% formic acid (B) were used as mobile phases. The LC flow rate was set at 0.4 mL/min. The binary linear gradient was initiated with 80% B and allowed to run for 0.5 min. Then gradient was increased to 100% B in 5 min. and hold for 5min. Then it was immediately switched to 80% B and re-equilibrated for 2 min before the next injection. The MS instrument was equipped with an electrospray chemical (ESI) ion source operating in the negative mode for FFAs analysis. For the determination of FFAs, ACQUITY UPLC® CSH™ C8 column (100X 2.1 mm; particle size 1.8 µm, Waters, Milford, MA) was used. Mobile phase A was 1 mM Ammonium Acetate in water and mobile phase B was 1 mM Ammonium Acetate in a mixture of Acetonitrile: MeOH (8:2). The binary pump was set to isocratic 90% B for eluting the free fatty acid. The m/z of FFA 18:0 and 16:0 were 233.2642, and 255.2328, respectively. Deuterated FFA 16:0-d31 with m/z 286.4273 was used as the internal standard. For the determination of LPCs, ESI ion source was switched to positive mode. The m/z of LPCs 16:0, 18:0, and 19:0 (IS) were monitored at 496.3394, 524.3719, and 538.3864 respectively. For determination of LPG, the same C18 column was used with 1 mM ammonium formate in water as mobile phase A and 1 mM ammonium formate in MeOH as mobile phase B. The binary linear gradient was initiated at 70% B and allowed to run for 0.5 min, increased to 100% until 4.6 min and maintained constant until 9 min. Then mobile phase was immediately changed back to 70%, and initial conditions established at 10 min. The analysis was performed with the ESI ion source in negative mode. The, m/z of LPG 18:0 and 16:0 (IS) were 511.3034, and 483.2727 respectively. The analytical conditions are summarized in the table below (Table 1.)
2.3 Preparation of calibration standards and quality control samples
100 µg/mL stock solution of FFA 16:0 and 18:0 mixture was used to prepare calibrators at 1, 5, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400 ng/mL with final 200 ng/mL FFA 16:0-d31 as the internal standard (IS). The continues calibration verification (CCV) samples were prepared at 20 ng/mL (low QC), 125 ng/mL (middle QC), and 375 ng/mL (high QC). The sample solvent was methanol for sample dilution.
For LPCs, 1.0 µg/mL stock solution of LPC 16:0, LPC 18:0 and LPG 18:0 were used to prepare three calibration plots with calibrators at concentrations of 10, 25, 50, 100, 150,200, 250, 300, 350, 400 ng/ mL, each calibrator contained 200 ng/mL LPC 19:0 and LPG 16:0 as internal standards (IS) for LPCs and LPG respectively. The CCV samples were prepared at 20 ng/mL (low QC), 225 ng/mL (middle QC), and 375 ng/mL (high QC). The sample solvent was methanol for reverse phase separation. The process was similar for both LPCs and LPG determinations.
2.4 Preparation of liposomal samples for LC-MS analysis
All stock solutions of liposomal formulations were prepared to equal 1000 µg/mL HSPC. Fifty µL of liquid liposomal formulations were dissolved in 429 mL of Chloroform/Methanol (1:1, v/v) mixture. For lyophilized powder products, 10 mg of sample was weighed using an analytical balance and suspended in 100 µL of MilliQ water and then re-dissolved in 1510 µL of Chloroform/Methanol (1:1, v/v) mixture. The disintegrated liposome sample was further diluted 200 times in methanol for LPCs and FFAs, and diluted 100X for LPG before analysis. The concentration of IS was maintained at 200 ng/mL.
2.5 Analytical method validation
The method validation was carried out as specified in USP 40-NF-39 <1225> validation compendial procedures (2015), the ICH (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use) guidance for method validation of analytical procedures (1996), and FDA guidance for industry on Bioanalytical Method Validation (2018).
2.5.1 Selectivity
All samples were freshly prepared from hydrogenated soybean phosphocholine (HSPC) powder at 1 mg/mL and DSPG at 0.4 mg/mL, which match HSPC and DSPG concentrations in the liposome drug products (LDPs) samples. The selected ion chromatograms for FFAs (16:0 and 18:0; 255.2328 and 283.2642), LPCs (16:0 and 18:0;496.3394, 524.3719) and LPG 18:0 (511.3034) were extracted from the total ion chromatogram using their respective m/z ratios.
2.5.2 Linearity
Calibration regression lines were calculated using a weighted linear regression model. The calibration plot was generated from the peak area ratio of analytes and internal standards. The ratios were then plotted on the y-axis against the concentration of analytes to generate the calibration plots.
2.5.3 Limit of detection (LOD) and the limit of quantitation (LOQ)
The sensitivity of the developed analytical procedure was evaluated by determining the limit of detection (LOD) and the limit of quantitation (LOQ). The lowest calibration standard with a signal-noise ratio (S/N) ≥ 3 was used as LOD criteria, and S/N ≥ 10 was used to establish LOQ.
2.5.4 Accuracy and precision
Method precision and accuracy were examined by processing the CCV standards in three continuous days at three concentrations (low, medium, high; n=5, each). The intra- day and inter-day precision were calculated as the percentage of relative standard deviation (RSD%) of the determined value. Accuracy was expressed as the mean calculated concentration in comparison with the theoretical concentration. The acceptance criteria were within ±15% for precision and 85-115% for accuracy.
3. Results and Discussion:
3.1 Stock solution preparation
To confirm that HSPC and DSPG raw materials do not contain degradation products, freshly made HSPC and DSPG stock solution was further diluted with mobile phase for LCMS analysis. HSPC is readily soluble in Chloroform/Methanol (1:1, v/v) at 1 mg/mL at room temperature. DSPG sodium salt is not soluble in Chloroform/Methanol (1:1, v/v) mixture at 0.1 mg/mL concentration. Various solvents were tested to dissolve DSPG at 0.5 mg/mL, including DMSO, chloroform, methanol, water, isopropanol, acetone, acetonitrile. Finally, a relatively complex solvent system was found to be a good combination to dissolve DSPG at a concentration of 0.5 mg/mL. This method involves first addition of acetone, acetonitrile, and isopropanol as one part each to make an emulsified DSPG. Then, three parts of chloroform were added to make a milky suspension followed by the addition of one part of water to make an emulsion. The final step involves breaking the emulsion by addition of three parts water to make 0.5 mg/mL DSPG stock solution.
Usually, DSPG can be dissolved in a common organic solvent by heating to 65°C (Richard T. Profitt, 1999). In our experiment, we were concerned that exposure of DSPG to an elevated 65°C may generate artifacts of degradation products, so we developed a room temperature solvent system to make the DSPG stock solution and avoid potential degradation of tested starting materials.
3.2 LC-MS method development
3.2.1 Internal standards
The ion intensity of an analyte measured with ESI-MS could be affected by many factors related to sample preparation, ionization conditions, tuning conditions, the analyzer and detector used in the mass spectrometer. Minor changes of these factors could lead to significant alterations in ion intensity from one condition to another (Wanget al., 2017). Therefore, for the accurate quantitation of trace amount of lipid degradation products in the LDPs formulations, an internal standard (IS) was added to samples of liposomal products before disintegration and analyzed at the same time as the sample. The best internal standards are radiolabeled compounds of target compounds; however, radiolabeled compounds are not available or expensive to synthesize. In this case, another compound of target compound class can be a good alternative for internal standard. In our study, odd carbon number LPC 19:0 was used as IS for quantitation of LPC 18:0. LPG 16:0 is selected as IS for LPG 18:0, since HSPC and DSPG hydrolyzed products should not contain LPG 16:0. FFA 16:0-d31 was used as IS for the quantitation of FFAs.
3.2.2 Q-TOF MS
Electrospray ionization (ESI) in positive mode is commonly used for LPC and PC analysis in LCMS because it can effectively ionize the choline head group of PCs (Cajka and Fiehn, 2014). However, negative ionization provides superior results for certain lipid classes, such as phosphatidylglycerol, phosphatidylserine, and phosphatidic acid (Seppanen-Laakso and Oresic, 2009). LPC can be detected under positive charge with a [M+H]+, while LPG and FFAs give a better sensitivity under negative mode with a [M- H].The Exact m/z of targeted compounds are listed in Table 1 (Table 2).
3.2.3 LC Separation
LC conditions for the separation and ionization efficiency were also investigated by using different mobile phase combinations and gradient with different analytical columns. There are different key excipients used in the liposome formulation including HSPC, cholesterol, DSPG, DSPE-mPEG, and antioxidants (tocopherol). The excipients are present in high quantity relative to impurities. Using direct injection analysis of liposome formulations, the critical step is to separate these PC hydrolyzed impurities from other lipids excipients. LPC and LPG with a single carbon chain are more polar than HSPC and DSPG. Under ACQUITY UPLC® CSH™ C18 column (100 X 2.1 mm; particle size 1.8 µm) column, these compounds can be baseline separated (Figure 2). When a C18 column was used for FFAs, it showed that strong retention, which causes low recovery and carry over due to the non-polar nature of FFAs. To lower the interaction, a C8 column was used for quantitation of FFAs (Figure 2).
Concentrations of LPC 16:0, LPC 18:0, and LPG 18:0 were 150 ng/mL in methanol. FFAs concentration was 100 ng/mL in methanol. Concentration of all internal standards (LPC 19:0, LPG16:0 and FFA 16:0-d31) was 200 ng/mL in methanol.
Addition of mobile phase additives is necessary to obtain well resolved, symmetric peaks in LC-MS mass spectrometry. Due to the positive charge of LPC molecules, they can be detected under positive mode. To facilitate ionization, 0.1% formic acid was added to the mobile phase along with 10 mM ammonium formate to reduce tailing. The cationic head groups of PCs tend to interact with the residual silanol groups in the stationary phase causing peak tailing and ion pair formation of silanol groups with ammonium ions prevent this interaction (Zhong et al., 2010). During the method development, it was observed that greater than 1mM of ammonium formate is necessary to keep peak symmetry. LPG was detected under negative mode as it is naturally a negatively charged molecule at neutral pH, therefore, relatively high pH will facilitate ionization, thus, mobile phases were kept free from acidic additives. The presence of an optimum concentration of ammonium formate helps for baseline separation of sn1 and sn2 regioisomers of LPG. It was observed that as the ammonium formate concentration increases, the separation of sn1 and sn2 regioisomers was not optimum but the peak intensity was high. Therefore, we tested different concentrations of volatile buffer and it was found that 1 mM ammonium formate concentration gives the optimal signal in terms of resolution and intensity.
FFA was analyzed under the negative mode, and the ionization efficiency was relatively low in the neutral or slightly acidic mobile phase. Peak intensity was dramatically reduced when formic acid and ammonium formate were present. Therefore, higher pKa ammonium acetate buffer (1 mM ammonium acetate) which has a high pKa value was employed to reduce tailing. Regarding the mobile phase solvents, asymmetric peaks were obtained when methanol was used.
Therefore, acetonitrile was also used in the mobile phase in order to obtain better peak intensity. Thus, the optimized solvent composition of acetonitrile:methanol was 8:2, which was utilized as a solvent B for the separation of FFAs.
Both LPCs and LPGs exist in two different regioisomeric forms as a result of intramolecular acyl migration. These regioisomers, sn1, and sn2 closely resemble their structures; which present challenges in discriminating each structural configurations (Dong et al., 2010). In this method, sn1 and sn2 isomer were baseline separated (Figure2). For LPC 16:0, sn1 and sn2 peaks were observed at 3.017 min and 2.785 min respectively whereas for LPC 18:0, peaks were at 3.993 min and 3.753 min for sn1 and sn2 respectively. In regard to LPG 18:0, the retention time for both sn1 and sn2 regioisomers was found to be 3.405 min and 3.28 min respectively.
3.3 LPCs and LPG regioisomers
As early as 1982, Plueckthun and Dennis demonstrated that LPC 16:0 is the equilibrium mixture consisting of approximately 90% of the 1-acyl-2-lyso isomers and 10% of the 2-acyl-1-lyso isomers. They also showed that the rate of the acyl migration was pH dependent, with a minimum of around pH 4 to pH 5 (Plueckthun and Dennis, 1982). High temperature, pH (above physiological) and chromatography on silicic acid or alumina columns is also known to increase the acyl migration (Sugasini and Subbaiah, 2017). But the isomerization can be minimized by storing the samples in acidic pH and by lowering the temperature (Kielbowicz et al., 2012; Plueckthun and Dennis, 1982). In order to accurately quantitate the LPCs and LPG impurities in the final LDPs, it is very important to control the sample condition to minimize this acyl migration during sample preparation and analysis.
The other option is to use the total integrated peak area for the calculation of LPCs and LPG concentrations in both analytes and standards.
In our LDP samples, HSPC and DSPG will be potentially hydrolyzed to LPCs or LPGs. Therefore, both LPCs and LPGs exist as regioisomers consisting of 2-acyl-1-lyso (sn2) and 1-acyl-2-lyso (sn1) forms (Scheme 02).
Under our analysis conditions, the sample solution was in neutral pH, and the sample analysis was done within 10 min. The sum of integration peak area of sn1 and sn2 isomer was used for the calculation. This is based on a hypothesis of sn1 and sn2 having the same ionization efficiency under ESI condition. To confirm that the ionization efficiency of both sn1 and sn2 isomers were similar, an experiment was designed and performed using different pH conditions at 11 (basic), 2 (acidic) and 7 (neutral) to treat real LDP samples which contain sn1 and sn2 isomers. We found that the total integration area was comparable between samples before and after acyl migration confirming that the ionization efficiency ofboth sn1 and sn2 isomers are similar; thus, the method can be used for the determination of isomers of LPCs and LPGs by using the sum of integrated peak area.
3.4 Method validation
3.4.1 Calibration curve and limits of detection and quantitation
For all five study analytes, weighted linear regression model was used for generating calibration plots. For LPC 16:0 and 18:0, LOD were 2 ng/mL and 2.1 ng/mL, respectively. LOQ for LPC 16:0 and 18:0 were 6.5 ng/mL and 7 ng/mL respectively. LOD and LOQ for LPG 18:0 were 3.3 ng/mL and 10 ng/mL, respectively. Linear range 10-400 ng/mL was selected for both LPCs and LPG, which has R2 >0.99. For FFA 16:0 and 18:0, LOD were 1.7 ng/mL and 1.0 ng/mL, respectively, LOQ were 5 ng/mL for both FFAs. Linear range 5-400 ng/mL was selected for both of FFAs and R2 >0.99. The equation for calibration plots and parameters is listed in Table 2.
3.4.2 Precision and accuracy
Precision and accuracy were investigated by analyzing the mixed ICV or CCV standards at three different concentration levels over three consecutive days. Inter-day precision and intra-day precision and relative standard deviation were less than 15% for all analytes (Table 3). Accuracy values were in the range of 85-115%, which was within the acceptable limits for this parameter.
3.5 Quantitation of phospholipids degradation products in raw materials and LDPs
In the manufacturing process, various processing steps have been employed including dehydration, lyophilization, heat treatment, and dialysis (Maherani et al., 2011; Mozafari, 2005; Zoghi et al., 2018). During these steps, phospholipids may be hydrolyzed, and various degradation products can be formed, including LPCs, LPGs, and FFAs (Scheme 01). LPCs are hydrolyzed products from HSPC (which is a mixture of DSPC and PSPC); therefore, LPC 16:0 and LPC 18:0 should be formed. LPG 18:0 should be generated when DSPG is hydrolyzed. FFAs were generated from both HSPC and DSPG, thus, FFA16:0 (Palmitic acid) and FFA 18:0 (Stearic acid) can be formed.
Using our validated method, four commercial LDPs were analyzed for LPC 16:0, 18:0, LPG 18:0, and FFA 16:0, and 18:0. HSPC and DSPG were analyzed as controls. The data indicated that the level of lipid degradation products from raw materials were below the limit of detection (Table 4).
For all the LDP samples, the major impurities are LPC 18:0 and FFA 18:0 and range from1.61 – 6.08% mol of HSPC. Impurities with 16:0 carbon chain (LPC 16:0 and FFA 16:0) is relatively much less than that of 18:0, and the calculated range is from 0 to 1.06 %mol of HSPC. There is much lower content of 16:0 carbon chain in the starting material, with the free fatty acids (FFA 16:0 and FFA 18:0) composition in the HSPC ratio is 11.4:88.6 (Avanti Polar Lipids Inc, 2019). The hydrolyzed impurities are much higher in LDP1 than other LDPs which may be due to the extreme pH conditions employed for manufacturing LDP1 (Richard T. Profitt, 1999). LPG 18:0 and FFA 18:0 have been detected in LDP1 as high as 4.52% mol and 5.76% mol of HSPC and DSPG, respectively. This implies that rigorous conditions such as acidic (pH between 1-3), and high temperature (about 65°C) can accelerate the hydrolysis of HSPC and DSPG.
4. Conclusion
An UPLC-APCI-QTOF method has been developed and validated for quantitation of phospholipid degradation products in liposomal drugs. LPC 18:0, LPG 18:0, FFA 18:0 were the major impurities present in liposomal drug products with minor LPC 16:0 and FFA 16:0. These degradation products were not detected in HSPC and DSPG raw materials. The validated method may be utilized to quantify phospholipid degradation related impurities present in LDP formulations to ensure the quality and safety of liposomal drug products.
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