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Characterization of polydisperse macrogols and macrogol-based excipients via HPLC and charged aerosol detection
Accepted Manuscript Title: Characterization of polydisperse macrogols and macrogol-based excipients via HPLC and charged aerosol detection Authors: Christiane Theiss, Ulrike Holzgrabe PII: DOI: Reference:
S0731-7085(18)31151-8 https://doi.org/10.1016/j.jpba.2018.07.043 PBA 12114
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
15-5-2018 20-7-2018 21-7-2018
Please cite this article as: Theiss C, Holzgrabe U, Characterization of polydisperse macrogols and macrogol-based excipients via HPLC and charged aerosol detection, Journal of Pharmaceutical and Biomedical Analysis (2018), https://doi.org/10.1016/j.jpba.2018.07.043 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Characterization of polydisperse macrogols and macrogolbased excipients via HPLC and charged aerosol detection
Christiane Theiss, Ulrike Holzgrabe*
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Institute for Pharmacy and Food Chemistry, University of Wuerzburg, Am Hubland, 97074 Wuerzburg, Germany
*To whom correspondence should be addressed:
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Prof. Dr. Ulrike Holzgrabe, Institute for Pharmacy and Food Chemistry, University of Wuerzburg, Am Hubland, DE-97074 Wuerzburg, Germany; Tel. +499313185461, Email: [email protected]
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A generic HPLC-CAD method to characterize macrogol-based emulsifiers was developed. 13 emulsifiers were separated for qualitative analysis of the oligomer distribution The excipients and their precursors can be separated simultaneously. The method is suitable to separate polymer macrogols from PEG 300 to PEG 3000. Fatty acids with more than 12 carbon atoms can be detected by CAD.
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Highlights
Abstract
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Macrogol-based emulsifiers and their respective precursor substances, i.e. macrogols (PEG), fatty acids (FA), and fatty alcohols (FAA), are widely used excipients which are usually
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characterized by a series of tests described within the European Pharmacopoeia (Ph. Eur.). Examples are bulk parameters such as the hydroxyl value, the peroxide value, and the determination of fatty acids composition by gas chromatography. The choice of tests depends on the emulsifier considered and its possible precursors. Though all methods are
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well established, most of them are time consuming and, in some cases, prone to errors and exhibit a low reproducibility. Here, an alternative and supplemental method was developed, using a HPLC-system coupled to a charged aerosol detector (CAD). Seven PEG samples, five saturated as well as two nonsaturated FA samples, and two FAA samples were analyzed. Together with these precursors, 13 macrogol-based emulsifiers of 3 different groups, i.e. macrogol ethers with FAA, macrogol esters with FA, and polysorbates, were successfully analyzed for oligomeric distribution and free precursor molecules in one run.
Abbreviations: Charged aerosol detector (CAD), evaporative light scattering detector (ELSD), polyethylene glycol (PEG), fatty acid (FA), fatty alcohol (FAA), ethylene glycol unit (EGU); fatty acid methyl ester (FAME), average molecular weight (ØMr)
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Keywords: polyethylene glycol, charged aerosol detection, O/W-emulsifiers, alcohol ethoxylates, polysorbates, macrogol stearate
1. Introduction
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Macrogols (polyethylene glycols, PEGs) and their related excipients such as polysorbates,
alcohol ethoxylates, and macrogol fatty acid esters, are widely used oil-in-water emulsifiers (o/w) in pharmaceutical and cosmetic industries. Furthermore, many additional applications, including their use as solubilizers, penetration enhancers, and vehicles for release-controlled
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drug delivery systems are known [1, 2]. Polydisperse PEGs are mixtures of polymers
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containing different chain lengths of ethylene oxide units with a variable size distribution, contrary to monodisperse PEGs which consists of a single chain length. The aforementioned
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groups of emulsifiers, i.e. polysorbates, alcohol ethoxylates, macrogol fatty acid esters,
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comprise of heterogeneous compounds as shown in Tab. 1 [2-7] as well as in Fig. 1. Based on this wide field of applications, it is important to provide eligible tests for ensuring the
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quality and content uniformity for different batches. For characterization of excipients, predominantly polymeric substances and natural product mixtures like fatty acids, the European Pharmacopoeia (Ph. Eur.) describes the measurement of different nominal values
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being executed according to conventional protocols. An important example is the hydroxyl value, which represents the total amount of hydroxy groups after esterification and back
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titration of the excessive esterification reagent [8]. For the group of macrogols this value serves for the determination of the molecular weight. The peroxide value, which is determined by reduction of peroxides with iodide and back titration of the produced iodine [9], represents the amount of degraded nonsaturated compounds which can be found for
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example in polysorbate 80 which mainly consists of oleic acid esters beside of other fatty acids. An important degradation process of nonsaturated compounds is the autoxidation [10]. For the determination of the general composition of fatty acids the Ph. Eur. describes a gas chromatographic method with preceding derivatization with methanol and a catalyzing reagent such as boron trifluoride or trimethylsulfonium hydroxide to give fatty acid methyl esters (FAMEs) [11, 12].
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However, it is necessary to have more than this panel of methods for the full characterization of polymeric excipients. All compendial protocols measure bulk parameters, resulting from the contribution of many components, and are very time consuming, prone to errors and exhibit a moderate reproducibility. Thus, alternative approaches offering the possibility for automation, e.g. liquid chromatography, and more detailed structural information are anticipated. In general, most excipients and polymeric compounds lack of suitable chromophores making it impossible to apply the widely established UV/vis detector, and thus, the analysis
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challenging. However, innovative detectors such as the charged aerosol detector (CAD)
have become commercially available enabling the detection of non- and semi-volatile compounds not bearing a suitable chromophore. Of note, direct detection with CAD is
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simpler and avoids errors that are often associated with pre- and post-column derivatization.
This significantly improves accuracy and reproducibility [13]. Recently, the CAD was applied for the characterization of various excipients not having a chromophore, i.e., particularly
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polysorbates in classical formulations [14-19], protein formulations including antibodies [2022], macrogol lauryl ether which is used as both excipient [23] and API (polidocanol [24, 25]),
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and fatty acids in general [26-29]. Hyphenation with size exclusion chromatography coupled
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with CAD has also been reported for analyzing other industrial polymers such as polyacrylic
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acid and polystyrene [30].
To best of our knowledge no attempt to develop a generic method being capable of simultaneously determining macrogols and their respective ethers and esters as well as their
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possible precursors was reported so far. Here, we aim for the development of a liquid chromatographic method being able to separate macrogol-based emulsifiers and their
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respective precursors simultaneously utilizing a commonly applied reversed phase C18-
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column and charged aerosol detection.
2. Materials and methods 2.1. Reagents and materials
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Macrogol 300
(PEG 300;
average
molecular
weight
of
ØMr = 285
–
315 g/mol)
and macrogol 2000 (PEG 2000; ØMr = 1800 – 2000 g/mol) were purchased from Carl Roth GmbH
(Karlsruhe,
macrogol 1500
Germany),
(PEG 1500;
macrogol 400
ØMr = 1400
–
(PEG 400;
1600 g/mol),
–
420 g/mol),
macrogol 3000
(PEG 3000;
ØMr = 380
ØMr = 2700 – 3300 g/mol) from Merck KGaA (Darmstadt, Germany), macrogol 600 (PEG 600; ØMr = 570 – 630 g/mol), and macrogol 1000 (PEG 1000; ØMr = 950 – 1050 g/mol) from Sigma-Aldrich (Taufkirchen, Germany). 3
All fatty acids, dodecanoic acid (lauric acid, C12 FA; 98%), tetradecanoic acid (myristic acid, C14 FA; ≥ 99%), hexadecenoic acid (palmitic acid, C16 FA; 99%), heptadecanoic acid (margaric acid, C17 FA; ≥ 98%), octadecanoic acid (stearic acid, C18 FA; 95%, type II), (9Z)octadec-9-enoic acid (oleic acid, C18:1 FA; ≥ 99%), and (9Z,12Z)-octadec-9,12-dienoic acid (linoleic acid, C18:2 FA; ≥ 99%)
were purchased from Sigma-Aldrich (Taufkirchen,
Germany), 1-hexadecanol (cetyl alcohol, C16 FAA; 98%) from Cognis GmbH (Düsseldorf, Germany), and cetyl stearyl alcohol (mixture of 1-hexadecanol and 1-octadecanol; C16/18 FAA) from Caelo (Caesar & Lorentz GmbH, Hilden, Germany).
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The alcohol ethoxylates Brij® 30, Brij® C10, Brij® S20, and Brij® O20 were purchased from Sigma-Aldrich (Taufkirchen, Germany), Brij® 52 from Croda Europe Ltd. (Yorkshire, United
Kingdom), Brij® 72 from ICI Surfactants (Middlesbrough, Cleveland, UK), and Thesit ® from
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Fagron GmbH & Co.KG (Barsbüttel, Germany). The macrogol fatty acid esters Myrj ® 45, Myrj® 52, and Myrj® 59 were from Croda Europe Ltd. (Yorkshire, United Kingdom), Cremophor® RH40 was purchased from Sigma-Aldrich (Taufkirchen, Germany). The
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polysorbates Tween® 20 and Tween® 80 were both purchased from Croda France (Chocques, France).
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Ultra-pure water (>18.2 MΩ) was delivered by a Milli-Q Synthesis System (Merck Millipore,
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Schwalbach, Germany). Methanol (HiPerSolv Chromanorm®) was purchased from VWR
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International (Darmstadt, Germany), acetonitrile (gradient grade) and formic acid (puriss. pa.
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for HPLC, 49 – 51%) from Sigma-Aldrich (Taufkirchen, Germany).
2.2. Preparation of samples and mobile phases
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0.1% (v/v) formic acid was added to ultra-pure water (mobile phase A) and acetonitrile (mobile phase B), respectively.
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Macrogols were individually dissolved (100 mg/10 mL; stock solutions) in ultra-pure water and diluted with ultra-pure water to a final concentration of 0.1 mg/mL (sample solution). A second concentration level of the stock solution of PEG 3000 was diluted with ultra-pure water to a concentration of 1.0 mg/mL. All fatty acids, alcohols and macrogol-based
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emulsifiers were dissolved in methanol (1000 mg/100 mL; stock solutions), except Myrj® 59 (1000 mg/10 mL; stock solution) which was dissolved in mobile phase B. The stock solutions of all fatty acids and alcohols were diluted to a final concentration of 0.1 mg/mL with methanol. The stock solutions of all emulsifiers except Myrj® 59 were diluted with methanol to a concentration of 1.0 mg/mL. The stock solution of Myrj® 59 was diluted to a final concentration of 20 mg/mL with mobile phase B.
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2.3. Preparing samples for reproducibility and specificity Fresh samples were prepared for reproducibility and specificity testing. All macrogols were dissolved in ultra-pure water (100 mg/10 mL; stock solutions) and diluted with ultra-pure water to the final concentration of 0.1 mg/mL. All fatty acids, fatty alcohols, and all emulsifiers except Myrj® 59 were dissolved in methanol (1000 mg/100 mL; stock solutions) and diluted with methanol to the final concentration of 0.1 mg/mL. Myrj® 59 was dissolved in mobile
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phase B (1000 mg/100 mL; stock solution) and diluted with mobile phase B to the final
2.4. Apparatus and chromatographic procedure: HPLC-CAD
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concentration of 0.1 mg/mL.
Measurements were carried out using an Agilent 1100 HPLC system (Waldbronn, Germany)
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equipped with an online degasser and a binary pump. A YMC-Pack Pro C18 (150 x 4.0 mm,
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3 µm particle size, YMC Europe GmbH, Dinslaken, Germany) analytical column was used. The column temperature was room temperature. The injection volume was 10 µL. A linear
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gradient elution was applied (conditions are shown in Tab. 2) followed by a column re-
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equilibration step of 15 min. The mobile phase flow rate was set to 1.0 mL/min. Detection was performed using an ESA Corona® charged aerosol detector (ThermoFisher,
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Courtaboeuf, France) which was linked with the HPLC system by a 0.25 mm internal diameter PEEK capillary and a 0.22 µm stainless steel inlet-frit. Highly pure nitrogen (99.9%) was produced by an ESA Nitrogen Generator (ThermoFisher, Courtaboeuf, France) and gas
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inlet pressure was set to 35.0 psi. Filter was set to “none” and the electric current range to 100 pA. Chromatograms were recorded and processed using the Agilent ChemStation®
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Software (Rev B.03.02).
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2.5. Apparatus and chromatographic procedure: HPLC-MS To verify the complete separation of the individual oligomers, the method was transferred to a LC-MS system using an Agilent 1100 HPLC system connected to a 6300 Series Ion Trap from Agilent (Waldbronn, Germany). The sample solutions of PEG 300 – 1500 (c = 0.1 mg/mL) were analyzed via electron spray ionization (ESI) in positive mode. Different target masses were set to include the full range of monomers. Only the timeslot relevant for macrogols was recorded (for details and all relevant LC-MS instrument settings see Tab. 3).
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3. Results and discussion 3.1. Method development Starting point for method development was the protocol previously reported by Brinz and Holzgrabe [31] who applied an HPLC-ELSD method for PEG characterization. They used a simple linear water/methanol-gradient and the Waters XTerra® RP18 (250 x 4.6 mm, 5 µm particle size; Milford, MA, USA) analytical column. This method was capable of separating
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PEGs up to an average molar mass of 1500 with acceptable resolution values. However, the method was not applied to PEGs of higher chain lengths. Separation was achieved within a
runtime of about 60 min which was due to the low flow rate of 0.5 mL/min. However, the
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viscosity of the water/methanol-gradient and the column dimension (L = 250 mm) produced a backpressure of about 160 bar which limited the flow rate to 0.5 ml/min due to the pressure limitations of the instrumentation used.
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In order to be able to optimize each substance class individually, the method development was carried out in three steps. Step 1 (hydrophilic part) aimed at enhancing of the separation
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of macrogols, step 2 (lipophilic part) dealt with FA and FAA, and step 3 finally connected both
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strategies in order to achieve the best separation for the respective emulsifiers.
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3.2. Step one: Separation of the macrogols
To lower the backpressure mentioned in section 3.1, methanol in mobile phase B was replaced with acetonitrile which allowed flow rates of 1.0 mL/min and thus, a shorter run time.
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The aim was to optimize the resolution RS between the single oligomer chains to baseline separation (RS > 1.4) or at least RS > 1 which might be obtained by a higher selectivity and a
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higher separation efficiency [32]. In order to increase the efficiency cf. [33-35] the YMC-Pack Pro, a RP-18 column with smaller particles (3 µm versus 5 µm), was applied. Additionally, the column used has a slightly higher carbon load (16% versus 15% of the first) which might be able to positively impact the number of theoretical plates N.
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Compared to the original method, the developed gradient was fragmented in three gradient and two plateau steps to target the different chain lengths stepwise. The first gradient elutes the lower chain lengths (e.g. PEG 300 and PEG 400), followed by a short plateau at 15% B to accumulate the more lipophilic higher chain lengths. Afterwards two linear gradients are performed consecutively to separate the medium-sized chain lengths (e.g. PEG 600) and the longer chain lengths (e.g. PEG 1000 to PEG 1500). This additionally improved the resolution particularly between species with medium and high chain lengths. Finally, the gradients 6
resulted in a second plateau (32.5% B) to slowly elute the high chain lengths (PEG 2000 to PEG 3000). Representative chromatograms are shown in Fig. 2. PEG 300, 400, 600, 1000, and 1500 (chain lengths n = 3 – 54), each being a mixture of PEGs which have the corresponding average molecular weight, were completely separated into their respective oligomers, which was confirmed by transferring the method to LC-MS analysis and confirming the molecular masses of the signals (for further information see “Supplementary material, Fig. A-1”). Of note, the oligomers appearing at the edge of the distribution pattern could only be detected by means of the LC-MS measurements, despite
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the injection volume was reduced to 1 and 2 µL, respectively. This is due to the often higher sensitivity of the ion trap for ionizable analytes compared to the CAD [36, 37].
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For macrogols having higher chain lengths (PEG 2000 and PEG 3000) a decrease in resolution with increasing chain length (n > 50) was observed; nevertheless, the respective oligomer distribution could be evaluated. These two PEGs were not analyzed with LC-MS
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due to the high molar masses which would exceed the tolerable mass range of the detector.
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3.2.1. Calculation of the theoretical mass-to-charge values (m/z)
Since many factors must be considered such as different charging states, i.e. single charging
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states (z = 1) which is more common for macrogols of shorter chain lengths and multiple charging states (z = 2; 3;…) which occurred more frequently with increasing chain lengths,
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and the possibility of adducts formation with molecules of the mobile phase, the calculation of the theoretical m/z was performed using an excel spreadsheet. A formula was generated using the molar masses of each component in variable amounts to allow the combination of
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different charging states and possible adducts. The calculation is given by following equation:
with n(EGU) = total chain length of PEG oligomer, n(W) = amount of water adducts, n(S) = amount of sodium adducts, n(A) = amount of acetonitrile adducts, n(F) = amount of
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formic acid adducts, and n(W)/ n(S)/ n(A)/ n(F) ≤ z. The respective molar masses are as follows: Mr(term.) = 18.02 g/mol (molar mass of the terminal groups of the PEGs), Mr(EGU) = 44.03 g/mol (molar mass of one ethylene glycol unit),
Mr(W) = 18.015 g/mol,
Mr(S) = 22.99 g/mol,
Mr(F) = 46.03 g/mol.
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Mr(A) = 41.05 g/mol,
and
3.3. Step two: Separation of fatty acids and respective alcohols Due to the better solubility, all FAs and FAAs were dissolved in methanol instead of acetonitrile. During method development, peak broadening and tailing was observed for all FAs, in particular due to dissociation of the carboxylic moiety at a neutral pH. The coexistence of both free acids and the carboxylates during the separation can cause the peak broadening effects. To shift the equilibrium to the protonated form (free acid) the mobile phases were
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acidified by using formic acid as modifier.
As shown in Fig. 3 all FAs and FAAs considered were separated, except C17 FA and
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C16 FAA. However, due to their natural and industrial production [38-40], these two substances are not expected to simultaneously appear in considerably high amounts in the macrogol-based emulsifiers studied.
FAs and FAAs having shorter chain lengths were also analyzed (C6 FA, C8 FA, C10 FA,
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C12 FAA), but could not be detected using CAD due to their volatility [41]. These volatile
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compounds, except for a few monographs (i.e. polysorbate 20), are not mentioned in the
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respective monographs of the substances considered and thus are not further discussed. The elution order was as expected, depending on the chain length and on the fact whether a
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molecule bears a carboxyl or a hydroxyl group. Thus, the alcohols eluted later than the acids: tR(C16 FA) = 64 min, tR(C16 FAA) = 70 min, tR(C18 FA) = 75 min, and tR(C18 FAA) = 90 min.
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The FAs eluted, according to chain lengths, between 50 min (C12 FA) and 75 min (C18 FA). Analogously, C16 FAA was eluted before C18 FAA.
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The nonsaturated FAs C18:1 FA ((9Z)-octadec-9-enoic acid) and C18:2 FA ((9Z,12Z)-9,12octadecadienoic acid) seemed to have a lower interaction with the C18 material than their saturated analogue which could be explained by the steric orientation of the (Z)-configured
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carbon chain of these molecules [19]. This hypothesis would also explain the shorter retention time of C18:2 FA (approx. 58 min) in comparison to C18: FA (approx. 65 min) and
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C18 FA (approx. 75 min).
3.4. Step 3: Separation of the macrogol-based emulsifiers All emulsifiers except M59 were dissolved in methanol to ensure a complete dissolution of potential free FAs and FAAs. M59 was dissolved in mobile phase B, due to a poor solubility of the substance in methanol and diluted with mobile phase B. M59 was diluted to a higher concentration level (20 mg/mL) than the other emulsifiers to compensate the higher 8
molecular weight of the free PEGs (average of 100 EGU). For a better comparability to the emulsifiers and observation of the higher chain lengths the reference substance PEG 3000 was diluted to a second concentration level of 1.0 mg/mL.
3.4.1. Alcohol ethoxylates Alcohol ethoxylates are ethers of fatty alcohols (FAAs) etherified with polyethylene glycol of a
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specified average molecular weight. The substances considered were lauryl (C12 FAA) ethers (i.e. B30 and Th), cetyl (C16 FAA) ethers (i.e. B52 and BC10), stearyl (C18 FAA) ethers (i.e. B72 and BS20), and oleyl (C18:1 FAA) ethers (i.e. BO20). The general
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compositions are shown in Tab. 1 and the basic structures are given in Fig. 1. All ethers can contain the free FAA and free PEGs.
Both lauryl ethoxylates B30 and Th can contain free C12 FAA which was not detected due to the volatility of the alcohol. B30 is C12 FAA etherified with an average of 4 EGU, while Th
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has a longer chain of an average of 9 EGU. In Fig. 4 both show a distribution pattern around
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49 – 52 min. Due to the polymeric macrogols used for etherification the resulting ethers show the same distribution which leads to the conclusion that this pattern must represent the
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higher chain length ethers. B30 showed a second distribution pattern near the theoretical
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retention range of C12 FAA (55 – 60 min) which might be ethers of shorter chain lengths and, thus, are more dominated by the lipophilic component. Both B30 and Th can contain free PEGs which are not clearly visible in Fig. 4, due to the scaling of the chromatographic
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overlay, but were found in the size range of approx. 8 – 16 EGU (reference PEG 400) for B30 and of approx. 10 – 35 EGU (reference PEG 600) for Th (for other scaling see
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“Supplemental material, Fig. B-1”).
Both cetyl ethoxylates B52 and BC10 can contain free C16 FAA which could be detected in
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the B52 sample. B52 is C16 FAA etherified with an average of 2 EGU which could be clearly identified in Fig. 4. In the time range of 70 – 75 min the free FAA eluted followed by the ethers. Due to the etherification with shorter PEGs the peak shape broadened but otherwise remained as plain single peaks not showing the typical polymeric distribution. With increasing
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average chain lengths such as in BC10 (average of 10 EGU) the polymeric distribution of PEGs dominates the ethers. Here, the ether distribution at 60 – 72 min covers the corresponding fatty alcohol. Both samples contain free PEGs which were found in the size range of approx. 10 – 35 EGU (reference PEG 600) for B52 and approx. 17 – 35 EGU (reference PEG 1000) for BC10 (for further information see “Supplemental material, Fig. B1”).
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Stearyl ethoxylates B72 and BS20 can contain free C18 FAA which could be detected in B72, whereas in BS20 no free FAA was found. B72 is C18 FAA etherified with an average of 2 EGU which could be clearly identified in Fig. 4. In the time range of 88 – 95 min the free FAA eluted followed by the ethers. Due to the etherification with shorter PEGs the peak shape broadened but otherwise remained as single peaks not showing the typical polymeric distribution. With increasing average chain lengths such as in BS20 (average of 20 EGU) the polymeric distribution of PEGs dominates the ethers. Here, the ether distribution eluted in a time range of 70 – 90 min before the retention time of the corresponding FAA (95 min). Both
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samples could contain free PEGs but only in B72 free PEGs in the size range of approx. 10 – 40 EGU (references PEG 600 and PEG 1000) were found in low amounts at retention times of 15 – 20 min (for further information see “Supplemental material, Fig. B-1”).
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The oleyl ethoxylate BO20 consists of oleyl alcohol (C18:1 FAA) etherified with an average of 20 EGU. The substance can contain free oleyl alcohol and free PEGs. The sample is also
dominated by the polymeric distribution of the etherified PEGs. Three distribution patterns
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were found. The main pattern eluted in the time range of 60 – 70 min are the presumable C18:1 FAA ethers. The free FAA could not be detected due to the dominant ether distribution
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coeluting in the same time slot. The earlier eluting ether group (52 – 57 min) might be
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degraded ethers, whereas the later eluting ether group eluted in the time range of C18 FAA ethers (72 – 85 min) which might be possible by-products. Free PEGs were found in a wide
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size range of approx. 3 - > 50 EGU (references PEG 600 to PEG 1500) (for further information see “Supplemental material, Fig. B-1”).
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In general, a correlation between average number of EGU, etherified FAA and retention behavior can be drawn from the results shown in Fig. 4 for ethers of the same FAA and
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variable amount of EGU the retention time decreases with increasing chain length, i.e. the ethers of BS20 (20 EGU, stearyl alcohol) having shorter retention time ranges than the ethers of B72 (2 EGU, stearyl alcohol). Vice versa, ethers with an equal average number of
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EGU, such as for example B52 (2 EGU, cetyl alcohol) and B72 (2 EGU, stearyl alcohol) show a retention behavior depending on the etherified alcohol: macrogol ethers with
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C16 FAA (B52) had shorter retention times than macrogol ethers with C18 FAA (B72).
3.4.2. Macrogol fatty acid esters Macrogol stearates (M45, M52, M59) are esters of stearic acid and polyethylene glycol of a nominal average molecular weight. The substances consist of different ester types, depending on the type of stearic acid used for ethoxylation. The Ph. Eur. specifies two main types of stearic acid, i.e. type I, containing 40.0 – 60.0% C18 FA and, in sum, a minimum of 10
90.0% C16 FA plus C18 FA, as well as type II consisting of 90.0 – 99.0% of C18 FA and, in sum, a minimum of 96.0% C16 FA plus C18 FA [42]. Type II was used as a reference in this study. All macrogol stearates may contain the free corresponding FAs and free PEGs. M45 contains stearic esters with an average of 8 EGU. Since two polymeric distribution patterns are eluted in the time ranges of 59 – 63 min (C16 FA esters) and 70 – 77 min (C18 FA esters) separately, it seems likely that type I stearic acid was used for esterification. As shown in Fig. 5 the esters dominate the co-eluting corresponding free FAs. Free PEGs were found in a molecular sizes range of approx. 5 – 17 EGU (references PEG 300 and
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PEG 400).
M52 consists of stearic esters with an average of 40 EGU. The M52 sample also shows two
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polymeric distribution patterns which are eluted in the time ranges of 50 – 57 min (C16 FA esters) and 60 – 70 min (C18 FA esters) separately, so it is again likely that type I stearic acid was used for esterification. The polymeric ester distributions eluted significantly before the expected retention time of the corresponding FA. However, no free FAs were present in
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M52. Free PEGs were found in a molecular sizes range of approx. 17 – 54 (reference
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PEG 1500).
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M59 contains stearic macrogol esters of an average of 100 EGU. Due to the higher chain lengths and the wider size distribution the esters are highly dominated by the hydrophilic
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polymeric macrogols which results in lower retention time ranges (approx. 25 – 38 min and 42 – 75 min). The free FAs C16 FA and C18 FA are clearly separated and detected. Free than PEG 3000).
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PEGs were located in a wide size range of approx. 8 - >100 (references PEG 1000 to more
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In RH40 the glycerol triesters of 12-hydroxystearic acid having an average of 40 EGU are more hydrophilic than the corresponding macrogol stearates due to an additional hydroxyl moiety. Additionally, steric effects, due to the branched triester structure (Fig. 6), might
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reduce interactions with the stationary phase. This resulted in much shorter retention times (18 – 23 min) of the polymeric ester distribution close to the free PEGs (Fig. 5). Free PEGs were found in a size range of approx. 13 – 30 EGU (references PEG 600 and PEG 1000). The prominent peak eluting at a retention time of about 45 min could not be identified. It
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might be the free 12-hydroxystearic acid or coeluting triglycerol esters, but this could not be reliably verified by LC-MS.
3.4.3. Polysorbates
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Polysorbates consist of ethoxylated sorbic acid with an average of 20 EGU, one of the PEG side chains esterified with the corresponding FA which are C12 FA for T20 and C18:1 FA for T80, respectively. Both samples can contain free FAs and PEGs. In Fig. 7, the ester distribution (45 – 60 min) of T20 is shown which is dominated by the polymeric character of the corresponding PEGs. Due to the semi-volatility of C12 FA no free FA was detected. Free PEGs in the size range of approx. 10 – 40 EGU (references PEG 600 and PEG 1000) were found.
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T80 is characterized by an ester distribution (50 – 63 min) dominated by the polymeric character of the corresponding PEGs. Here, the free C18:1 FA was clearly separated from
the ester fraction and detected at a retention time of 65 min. Free PEGs were located in a
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size range of approx. 10 – 40 EGU (references PEG 600 and PEG 1000).
3.5. Summary of method characteristics
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3.5.1. Reproducibility
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For reproducibility triplicate measurements of all 0.1 mg/mL samples were performed. Each
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injection of the triplicate measurements was executed on consecutive days to ensure inter-
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day reproducibility. The aim of reducing the sample concentration was to produce a comparable level of all samples and to examine recovery of each sample.
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All single reference substances (PEG, FA, FAA) were easily recovered and retention times were reproduced during the triplicate measurements; thus, the different substances could be distinguished and identified. The main distribution of the emulsifiers was recovered as well as
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the free PEGs which is shown for M45 as example substance in Fig. 8. Here, in all 4 samples, free PEGs are detected in a size range of PEG 300 to PEG 400 such as the
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polymeric ester distributions of C16 FA esters (59 – 63 min) and C18 FA esters (70 – 77 min). Both, the distribution of the macrogol polymers and the distribution of the esters could be reproduced.
As a negative result some of the possible by-products in macrogol stearate, such as esters of
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other FA, e.g. C12 FA or C14 FA, were not detected within the test solutions (1) – (3), due to the lower concentration levels of 0.1 mg/mL and therefore, a lower sensitivity. In the reference solution (R; c = 1.0 mg/mL) these by-products were detected (Fig. 8). For the other emulsifiers studied, results were comparable.
12
3.5.2. Specificity Specificity was obtained for all reference substances (PEGs, FAs, and FAAs) which were clearly separated from each other, whereas some of the esters and ethers coeluted with the free precursor substances (e.g. BO20, M45). However, reducing the sample concentrations and, thus, reducing potential concentration-correlated peak-broadening effects, did not reduce the coelution. Hence, the specificity does not benefit from the reduction of sample
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concentration.
3.5.3. Sample preparation
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Considering the results of reproducibility (section 3.5.1) and specificity (section 3.1.2) sample concentrations are recommended as 0.1 mg/mL for references (PEGs, FAs, FAAs) and at least 1.0 mg/mL for emulsifiers to ensure the detectability of potential by-products and free
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precursor substances.
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4. Conclusion
The study describes a new HPLC-CAD method suitable for characterizing polymeric
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excipients primarily based on macrogols. It can be considered as a supplement to the established methods described in the Ph. Eur. and allows reducing the analytical efforts.
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Additionally, the method provides a much better reproducibility and batch-to-batch monitoring, especially for the distribution of the oligomers and the detection of the individual components, than the determination of bulk parameters. Free macrogols could be completely
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separated into the single oligomers (n = 3 – 54). In some cases, free fatty acids and alcohols coeluted with the respective esters and ethers, whereas in other cases they were completely
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separated. Furthermore, the distribution of ester and ether oligomers could be evaluated for the first time.
Current protocols consider single components only, e.g. determining the amount of free fatty
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acids and the fatty acid composition of polysorbates after saponification [19], the behavior of polysorbate under specific conditions, e.g. gastrointestinal hydrolysis [15] and oxidative degradation [18], and the distribution of polymeric macrogols [31]. The simultaneous determination of free macrogols, fatty alcohols and acids, and the respective ethers and esters using HPLC-CAD was not reported so far. In contrast to the established pharmacopoeial methods which are highly time-consuming and prone to errors, the described method is more comfortable and accurate. Whilst for adhering 13
to protocols such as determining the hydroxyl value the continuous presence of an analyst must be ensured, the HPLC-CAD method is able to run autonomously after sample preparation is done and thus can easily be automated. Furthermore, the method is compatible with LC-MS, as long as the respective molar masses are within the tolerable mass range of the instrumentation used. This enables identifying impurities which are produced during the production process or monitoring degradation
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products which is an important aspect during long term stability assessments.
Conflict of interest
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None of the authors stated a conflict of interest.
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[38]
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Tables Table 1 Nominal composition of the analyzed macrogol-based emulsifiers and internal labeling. The internal labeling is used in Section 3. Only the main components are mentioned (EGU = ethylene glycol unit). Internal labeling1
Composition (suppliers’ information)/ further classification
Brij® 30
B30
average of 4 EGU etherified with lauryl alcohol [6]
Brij® 52
B52
average of 2 EGU etherified with cetyl alcohol [3]
Brij® 72
B72
average of 2 EGU etherified with stearyl alcohol [4]
Brij® C10
BC10
average of 10 EGU etherified with cetyl alcohol [2]
Brij® S20
BS20
average of 20 EGU etherified with stearyl alcohol [2]
Brij® O20
BO20
average of 20 EGU etherified with oleyl alcohol [2]
Polidocanol, Thesit®
Th
Cremophor® RH40
RH40
Polysorbate 80, Tween® 80
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A
average of 40 EGU esterified with 12hydroxystearic acid and glycerol [2]
average of 40 EGU esterified with stearic acid [2]
M59
average of 100 EGU esterified with stearic acid [2]
T20
sorbitol anhydrides copolymerized with about 20 EGU, esterified with mainly lauric acid [2]
T80
sorbitol anhydrides copolymerized with about 20 EGU, esterified with mainly oleic acid [2]
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M52
A
Polysorbate 20, Tween® 20
average of 9 EGU etherified with lauryl alcohol [5]
average of 8 EGU esterified with stearic acid [2]
M45
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Myrj® 59
M
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Myrj® 45 Myrj® 52
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Substance
1
Internal labeling was assigned as follows: initial letter of the registered trade name (“B” for Brij®. “M” for Myrj®, “T” for Tween®) followed by the type number (i.e. Brij ® C10 as BC10). The two outstanding samples Thesit (“Th”) and Cremophor® RH40 (“RH40”) are labelled differently.
19
B [%]
0
10
1
10
6
15
7.5
15
17.5
30
27.5
32.5
35
32.5
45
75
50
80
70
85
105
85
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t [min]
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Table 2 Chromatographic conditions: time table of the linear gradient for the HPLCCAD method (flow rate = 1 mL/min; injection volume: 10 µL).
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Table 3 Chromatographic conditions for LC-MS measurements. Different settings (1,2) for ion trap were used to receive different charging levels of the analytes. Flow rate
1.0 mL/min 1 µL 2 µL
PEG 300, PEG 400, PEG 600 PEG 1000, PEG 1500
Target masses
PEG 300 PEG 400 PEG 600 PEG 1000 PEG 1500
283; 415 371; 547 459; 679; 899 723; 899; 1119; 1339 1207; 1427; 1648; 1868
Setting (1)
stability nebulizer drying gas (nitrogen flow) drying temp.
20% 30.0 psi 8.00 L/min
stability nebulizer drying gas (nitrogen flow) drying temp.
100% 50.0 psi 10.00 L/min
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Gradient
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350 °C
M
t [min] 0 1 6 7.5 17.5 27.5 35
350 °C
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Setting (2)
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column re-equilibration: 10 min
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Injection volume
B [%] 10 10 15 15 30 32.5 32.5
Figures
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Fig. 1 General structures of macrogol-based emulsifiers and the analyzed precursor molecules (macrogols, FA and FAA). n/a: reference substances were not available.
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Fig. 2 Separation of different PEGs using HPLC-CAD. Sample concentration for all: 0.1 mg/mL.
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Fig. 3 Separation of a mix containing 0.1 mg/mL of each fatty acid (FA: C12, C14, C16, C17, C18, C18:1, C18:2) and fatty alcohol (FAA: C16, C16/18).
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Fig. 4 Separation results of alcohol ethoxylates. For PEG 3000 a sample concentration of 1 mg/mL sample was used. “Mix” = mixed sample of FA: C12, C16, C17, C18, C18:1, C18:2 and FAA: C16, C16/18.
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Fig. 5 Separation results of macrogol fatty acid esters. For PEG 3000 a sample concentration of 1 mg/mL sample was used. “Mix” = mixed sample of FA: C12, C16, C17, C18, C18:1, C18:2 and FAA: C16, C16/18.
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Fig. 6 Structure of Cremophor® RH40.
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Fig. 7 Separation results of polysorbates. For PEG 3000 a sample concentration of 1 mg/mL sample was used. “Mix” = mixed sample of FA: C12, C16, C17, C18, C18:1, C18:2 and FAA: C16, C16/18.
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Fig. 8 Overlayed chromatograms of triplicate measurements (1) – (3) and reference runs (R) of M45 and the reference substances PEG 400 and C18 FA, respectively. Zoomed area of the relevant retention range (PEGs, FAs, FAAs, esters).
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S0731-7085(18)31151-8 https://doi.org/10.1016/j.jpba.2018.07.043 PBA 12114
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
15-5-2018 20-7-2018 21-7-2018
Please cite this article as: Theiss C, Holzgrabe U, Characterization of polydisperse macrogols and macrogol-based excipients via HPLC and charged aerosol detection, Journal of Pharmaceutical and Biomedical Analysis (2018), https://doi.org/10.1016/j.jpba.2018.07.043 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Characterization of polydisperse macrogols and macrogolbased excipients via HPLC and charged aerosol detection
Christiane Theiss, Ulrike Holzgrabe*
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Institute for Pharmacy and Food Chemistry, University of Wuerzburg, Am Hubland, 97074 Wuerzburg, Germany
*To whom correspondence should be addressed:
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Prof. Dr. Ulrike Holzgrabe, Institute for Pharmacy and Food Chemistry, University of Wuerzburg, Am Hubland, DE-97074 Wuerzburg, Germany; Tel. +499313185461, Email: [email protected]
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A generic HPLC-CAD method to characterize macrogol-based emulsifiers was developed. 13 emulsifiers were separated for qualitative analysis of the oligomer distribution The excipients and their precursors can be separated simultaneously. The method is suitable to separate polymer macrogols from PEG 300 to PEG 3000. Fatty acids with more than 12 carbon atoms can be detected by CAD.
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Highlights
Abstract
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Macrogol-based emulsifiers and their respective precursor substances, i.e. macrogols (PEG), fatty acids (FA), and fatty alcohols (FAA), are widely used excipients which are usually
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characterized by a series of tests described within the European Pharmacopoeia (Ph. Eur.). Examples are bulk parameters such as the hydroxyl value, the peroxide value, and the determination of fatty acids composition by gas chromatography. The choice of tests depends on the emulsifier considered and its possible precursors. Though all methods are
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well established, most of them are time consuming and, in some cases, prone to errors and exhibit a low reproducibility. Here, an alternative and supplemental method was developed, using a HPLC-system coupled to a charged aerosol detector (CAD). Seven PEG samples, five saturated as well as two nonsaturated FA samples, and two FAA samples were analyzed. Together with these precursors, 13 macrogol-based emulsifiers of 3 different groups, i.e. macrogol ethers with FAA, macrogol esters with FA, and polysorbates, were successfully analyzed for oligomeric distribution and free precursor molecules in one run.
Abbreviations: Charged aerosol detector (CAD), evaporative light scattering detector (ELSD), polyethylene glycol (PEG), fatty acid (FA), fatty alcohol (FAA), ethylene glycol unit (EGU); fatty acid methyl ester (FAME), average molecular weight (ØMr)
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Keywords: polyethylene glycol, charged aerosol detection, O/W-emulsifiers, alcohol ethoxylates, polysorbates, macrogol stearate
1. Introduction
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Macrogols (polyethylene glycols, PEGs) and their related excipients such as polysorbates,
alcohol ethoxylates, and macrogol fatty acid esters, are widely used oil-in-water emulsifiers (o/w) in pharmaceutical and cosmetic industries. Furthermore, many additional applications, including their use as solubilizers, penetration enhancers, and vehicles for release-controlled
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drug delivery systems are known [1, 2]. Polydisperse PEGs are mixtures of polymers
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containing different chain lengths of ethylene oxide units with a variable size distribution, contrary to monodisperse PEGs which consists of a single chain length. The aforementioned
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groups of emulsifiers, i.e. polysorbates, alcohol ethoxylates, macrogol fatty acid esters,
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comprise of heterogeneous compounds as shown in Tab. 1 [2-7] as well as in Fig. 1. Based on this wide field of applications, it is important to provide eligible tests for ensuring the
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quality and content uniformity for different batches. For characterization of excipients, predominantly polymeric substances and natural product mixtures like fatty acids, the European Pharmacopoeia (Ph. Eur.) describes the measurement of different nominal values
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being executed according to conventional protocols. An important example is the hydroxyl value, which represents the total amount of hydroxy groups after esterification and back
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titration of the excessive esterification reagent [8]. For the group of macrogols this value serves for the determination of the molecular weight. The peroxide value, which is determined by reduction of peroxides with iodide and back titration of the produced iodine [9], represents the amount of degraded nonsaturated compounds which can be found for
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example in polysorbate 80 which mainly consists of oleic acid esters beside of other fatty acids. An important degradation process of nonsaturated compounds is the autoxidation [10]. For the determination of the general composition of fatty acids the Ph. Eur. describes a gas chromatographic method with preceding derivatization with methanol and a catalyzing reagent such as boron trifluoride or trimethylsulfonium hydroxide to give fatty acid methyl esters (FAMEs) [11, 12].
2
However, it is necessary to have more than this panel of methods for the full characterization of polymeric excipients. All compendial protocols measure bulk parameters, resulting from the contribution of many components, and are very time consuming, prone to errors and exhibit a moderate reproducibility. Thus, alternative approaches offering the possibility for automation, e.g. liquid chromatography, and more detailed structural information are anticipated. In general, most excipients and polymeric compounds lack of suitable chromophores making it impossible to apply the widely established UV/vis detector, and thus, the analysis
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challenging. However, innovative detectors such as the charged aerosol detector (CAD)
have become commercially available enabling the detection of non- and semi-volatile compounds not bearing a suitable chromophore. Of note, direct detection with CAD is
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simpler and avoids errors that are often associated with pre- and post-column derivatization.
This significantly improves accuracy and reproducibility [13]. Recently, the CAD was applied for the characterization of various excipients not having a chromophore, i.e., particularly
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polysorbates in classical formulations [14-19], protein formulations including antibodies [2022], macrogol lauryl ether which is used as both excipient [23] and API (polidocanol [24, 25]),
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and fatty acids in general [26-29]. Hyphenation with size exclusion chromatography coupled
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with CAD has also been reported for analyzing other industrial polymers such as polyacrylic
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acid and polystyrene [30].
To best of our knowledge no attempt to develop a generic method being capable of simultaneously determining macrogols and their respective ethers and esters as well as their
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possible precursors was reported so far. Here, we aim for the development of a liquid chromatographic method being able to separate macrogol-based emulsifiers and their
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respective precursors simultaneously utilizing a commonly applied reversed phase C18-
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column and charged aerosol detection.
2. Materials and methods 2.1. Reagents and materials
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Macrogol 300
(PEG 300;
average
molecular
weight
of
ØMr = 285
–
315 g/mol)
and macrogol 2000 (PEG 2000; ØMr = 1800 – 2000 g/mol) were purchased from Carl Roth GmbH
(Karlsruhe,
macrogol 1500
Germany),
(PEG 1500;
macrogol 400
ØMr = 1400
–
(PEG 400;
1600 g/mol),
–
420 g/mol),
macrogol 3000
(PEG 3000;
ØMr = 380
ØMr = 2700 – 3300 g/mol) from Merck KGaA (Darmstadt, Germany), macrogol 600 (PEG 600; ØMr = 570 – 630 g/mol), and macrogol 1000 (PEG 1000; ØMr = 950 – 1050 g/mol) from Sigma-Aldrich (Taufkirchen, Germany). 3
All fatty acids, dodecanoic acid (lauric acid, C12 FA; 98%), tetradecanoic acid (myristic acid, C14 FA; ≥ 99%), hexadecenoic acid (palmitic acid, C16 FA; 99%), heptadecanoic acid (margaric acid, C17 FA; ≥ 98%), octadecanoic acid (stearic acid, C18 FA; 95%, type II), (9Z)octadec-9-enoic acid (oleic acid, C18:1 FA; ≥ 99%), and (9Z,12Z)-octadec-9,12-dienoic acid (linoleic acid, C18:2 FA; ≥ 99%)
were purchased from Sigma-Aldrich (Taufkirchen,
Germany), 1-hexadecanol (cetyl alcohol, C16 FAA; 98%) from Cognis GmbH (Düsseldorf, Germany), and cetyl stearyl alcohol (mixture of 1-hexadecanol and 1-octadecanol; C16/18 FAA) from Caelo (Caesar & Lorentz GmbH, Hilden, Germany).
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The alcohol ethoxylates Brij® 30, Brij® C10, Brij® S20, and Brij® O20 were purchased from Sigma-Aldrich (Taufkirchen, Germany), Brij® 52 from Croda Europe Ltd. (Yorkshire, United
Kingdom), Brij® 72 from ICI Surfactants (Middlesbrough, Cleveland, UK), and Thesit ® from
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Fagron GmbH & Co.KG (Barsbüttel, Germany). The macrogol fatty acid esters Myrj ® 45, Myrj® 52, and Myrj® 59 were from Croda Europe Ltd. (Yorkshire, United Kingdom), Cremophor® RH40 was purchased from Sigma-Aldrich (Taufkirchen, Germany). The
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polysorbates Tween® 20 and Tween® 80 were both purchased from Croda France (Chocques, France).
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Ultra-pure water (>18.2 MΩ) was delivered by a Milli-Q Synthesis System (Merck Millipore,
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Schwalbach, Germany). Methanol (HiPerSolv Chromanorm®) was purchased from VWR
M
International (Darmstadt, Germany), acetonitrile (gradient grade) and formic acid (puriss. pa.
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for HPLC, 49 – 51%) from Sigma-Aldrich (Taufkirchen, Germany).
2.2. Preparation of samples and mobile phases
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0.1% (v/v) formic acid was added to ultra-pure water (mobile phase A) and acetonitrile (mobile phase B), respectively.
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Macrogols were individually dissolved (100 mg/10 mL; stock solutions) in ultra-pure water and diluted with ultra-pure water to a final concentration of 0.1 mg/mL (sample solution). A second concentration level of the stock solution of PEG 3000 was diluted with ultra-pure water to a concentration of 1.0 mg/mL. All fatty acids, alcohols and macrogol-based
A
emulsifiers were dissolved in methanol (1000 mg/100 mL; stock solutions), except Myrj® 59 (1000 mg/10 mL; stock solution) which was dissolved in mobile phase B. The stock solutions of all fatty acids and alcohols were diluted to a final concentration of 0.1 mg/mL with methanol. The stock solutions of all emulsifiers except Myrj® 59 were diluted with methanol to a concentration of 1.0 mg/mL. The stock solution of Myrj® 59 was diluted to a final concentration of 20 mg/mL with mobile phase B.
4
2.3. Preparing samples for reproducibility and specificity Fresh samples were prepared for reproducibility and specificity testing. All macrogols were dissolved in ultra-pure water (100 mg/10 mL; stock solutions) and diluted with ultra-pure water to the final concentration of 0.1 mg/mL. All fatty acids, fatty alcohols, and all emulsifiers except Myrj® 59 were dissolved in methanol (1000 mg/100 mL; stock solutions) and diluted with methanol to the final concentration of 0.1 mg/mL. Myrj® 59 was dissolved in mobile
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phase B (1000 mg/100 mL; stock solution) and diluted with mobile phase B to the final
2.4. Apparatus and chromatographic procedure: HPLC-CAD
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concentration of 0.1 mg/mL.
Measurements were carried out using an Agilent 1100 HPLC system (Waldbronn, Germany)
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equipped with an online degasser and a binary pump. A YMC-Pack Pro C18 (150 x 4.0 mm,
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3 µm particle size, YMC Europe GmbH, Dinslaken, Germany) analytical column was used. The column temperature was room temperature. The injection volume was 10 µL. A linear
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gradient elution was applied (conditions are shown in Tab. 2) followed by a column re-
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equilibration step of 15 min. The mobile phase flow rate was set to 1.0 mL/min. Detection was performed using an ESA Corona® charged aerosol detector (ThermoFisher,
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Courtaboeuf, France) which was linked with the HPLC system by a 0.25 mm internal diameter PEEK capillary and a 0.22 µm stainless steel inlet-frit. Highly pure nitrogen (99.9%) was produced by an ESA Nitrogen Generator (ThermoFisher, Courtaboeuf, France) and gas
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inlet pressure was set to 35.0 psi. Filter was set to “none” and the electric current range to 100 pA. Chromatograms were recorded and processed using the Agilent ChemStation®
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Software (Rev B.03.02).
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2.5. Apparatus and chromatographic procedure: HPLC-MS To verify the complete separation of the individual oligomers, the method was transferred to a LC-MS system using an Agilent 1100 HPLC system connected to a 6300 Series Ion Trap from Agilent (Waldbronn, Germany). The sample solutions of PEG 300 – 1500 (c = 0.1 mg/mL) were analyzed via electron spray ionization (ESI) in positive mode. Different target masses were set to include the full range of monomers. Only the timeslot relevant for macrogols was recorded (for details and all relevant LC-MS instrument settings see Tab. 3).
5
3. Results and discussion 3.1. Method development Starting point for method development was the protocol previously reported by Brinz and Holzgrabe [31] who applied an HPLC-ELSD method for PEG characterization. They used a simple linear water/methanol-gradient and the Waters XTerra® RP18 (250 x 4.6 mm, 5 µm particle size; Milford, MA, USA) analytical column. This method was capable of separating
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PEGs up to an average molar mass of 1500 with acceptable resolution values. However, the method was not applied to PEGs of higher chain lengths. Separation was achieved within a
runtime of about 60 min which was due to the low flow rate of 0.5 mL/min. However, the
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viscosity of the water/methanol-gradient and the column dimension (L = 250 mm) produced a backpressure of about 160 bar which limited the flow rate to 0.5 ml/min due to the pressure limitations of the instrumentation used.
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In order to be able to optimize each substance class individually, the method development was carried out in three steps. Step 1 (hydrophilic part) aimed at enhancing of the separation
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of macrogols, step 2 (lipophilic part) dealt with FA and FAA, and step 3 finally connected both
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A
strategies in order to achieve the best separation for the respective emulsifiers.
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3.2. Step one: Separation of the macrogols
To lower the backpressure mentioned in section 3.1, methanol in mobile phase B was replaced with acetonitrile which allowed flow rates of 1.0 mL/min and thus, a shorter run time.
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The aim was to optimize the resolution RS between the single oligomer chains to baseline separation (RS > 1.4) or at least RS > 1 which might be obtained by a higher selectivity and a
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higher separation efficiency [32]. In order to increase the efficiency cf. [33-35] the YMC-Pack Pro, a RP-18 column with smaller particles (3 µm versus 5 µm), was applied. Additionally, the column used has a slightly higher carbon load (16% versus 15% of the first) which might be able to positively impact the number of theoretical plates N.
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Compared to the original method, the developed gradient was fragmented in three gradient and two plateau steps to target the different chain lengths stepwise. The first gradient elutes the lower chain lengths (e.g. PEG 300 and PEG 400), followed by a short plateau at 15% B to accumulate the more lipophilic higher chain lengths. Afterwards two linear gradients are performed consecutively to separate the medium-sized chain lengths (e.g. PEG 600) and the longer chain lengths (e.g. PEG 1000 to PEG 1500). This additionally improved the resolution particularly between species with medium and high chain lengths. Finally, the gradients 6
resulted in a second plateau (32.5% B) to slowly elute the high chain lengths (PEG 2000 to PEG 3000). Representative chromatograms are shown in Fig. 2. PEG 300, 400, 600, 1000, and 1500 (chain lengths n = 3 – 54), each being a mixture of PEGs which have the corresponding average molecular weight, were completely separated into their respective oligomers, which was confirmed by transferring the method to LC-MS analysis and confirming the molecular masses of the signals (for further information see “Supplementary material, Fig. A-1”). Of note, the oligomers appearing at the edge of the distribution pattern could only be detected by means of the LC-MS measurements, despite
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the injection volume was reduced to 1 and 2 µL, respectively. This is due to the often higher sensitivity of the ion trap for ionizable analytes compared to the CAD [36, 37].
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For macrogols having higher chain lengths (PEG 2000 and PEG 3000) a decrease in resolution with increasing chain length (n > 50) was observed; nevertheless, the respective oligomer distribution could be evaluated. These two PEGs were not analyzed with LC-MS
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due to the high molar masses which would exceed the tolerable mass range of the detector.
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3.2.1. Calculation of the theoretical mass-to-charge values (m/z)
Since many factors must be considered such as different charging states, i.e. single charging
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states (z = 1) which is more common for macrogols of shorter chain lengths and multiple charging states (z = 2; 3;…) which occurred more frequently with increasing chain lengths,
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and the possibility of adducts formation with molecules of the mobile phase, the calculation of the theoretical m/z was performed using an excel spreadsheet. A formula was generated using the molar masses of each component in variable amounts to allow the combination of
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different charging states and possible adducts. The calculation is given by following equation:
with n(EGU) = total chain length of PEG oligomer, n(W) = amount of water adducts, n(S) = amount of sodium adducts, n(A) = amount of acetonitrile adducts, n(F) = amount of
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formic acid adducts, and n(W)/ n(S)/ n(A)/ n(F) ≤ z. The respective molar masses are as follows: Mr(term.) = 18.02 g/mol (molar mass of the terminal groups of the PEGs), Mr(EGU) = 44.03 g/mol (molar mass of one ethylene glycol unit),
Mr(W) = 18.015 g/mol,
Mr(S) = 22.99 g/mol,
Mr(F) = 46.03 g/mol.
7
Mr(A) = 41.05 g/mol,
and
3.3. Step two: Separation of fatty acids and respective alcohols Due to the better solubility, all FAs and FAAs were dissolved in methanol instead of acetonitrile. During method development, peak broadening and tailing was observed for all FAs, in particular due to dissociation of the carboxylic moiety at a neutral pH. The coexistence of both free acids and the carboxylates during the separation can cause the peak broadening effects. To shift the equilibrium to the protonated form (free acid) the mobile phases were
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acidified by using formic acid as modifier.
As shown in Fig. 3 all FAs and FAAs considered were separated, except C17 FA and
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C16 FAA. However, due to their natural and industrial production [38-40], these two substances are not expected to simultaneously appear in considerably high amounts in the macrogol-based emulsifiers studied.
FAs and FAAs having shorter chain lengths were also analyzed (C6 FA, C8 FA, C10 FA,
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C12 FAA), but could not be detected using CAD due to their volatility [41]. These volatile
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compounds, except for a few monographs (i.e. polysorbate 20), are not mentioned in the
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respective monographs of the substances considered and thus are not further discussed. The elution order was as expected, depending on the chain length and on the fact whether a
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molecule bears a carboxyl or a hydroxyl group. Thus, the alcohols eluted later than the acids: tR(C16 FA) = 64 min, tR(C16 FAA) = 70 min, tR(C18 FA) = 75 min, and tR(C18 FAA) = 90 min.
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The FAs eluted, according to chain lengths, between 50 min (C12 FA) and 75 min (C18 FA). Analogously, C16 FAA was eluted before C18 FAA.
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The nonsaturated FAs C18:1 FA ((9Z)-octadec-9-enoic acid) and C18:2 FA ((9Z,12Z)-9,12octadecadienoic acid) seemed to have a lower interaction with the C18 material than their saturated analogue which could be explained by the steric orientation of the (Z)-configured
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carbon chain of these molecules [19]. This hypothesis would also explain the shorter retention time of C18:2 FA (approx. 58 min) in comparison to C18: FA (approx. 65 min) and
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C18 FA (approx. 75 min).
3.4. Step 3: Separation of the macrogol-based emulsifiers All emulsifiers except M59 were dissolved in methanol to ensure a complete dissolution of potential free FAs and FAAs. M59 was dissolved in mobile phase B, due to a poor solubility of the substance in methanol and diluted with mobile phase B. M59 was diluted to a higher concentration level (20 mg/mL) than the other emulsifiers to compensate the higher 8
molecular weight of the free PEGs (average of 100 EGU). For a better comparability to the emulsifiers and observation of the higher chain lengths the reference substance PEG 3000 was diluted to a second concentration level of 1.0 mg/mL.
3.4.1. Alcohol ethoxylates Alcohol ethoxylates are ethers of fatty alcohols (FAAs) etherified with polyethylene glycol of a
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specified average molecular weight. The substances considered were lauryl (C12 FAA) ethers (i.e. B30 and Th), cetyl (C16 FAA) ethers (i.e. B52 and BC10), stearyl (C18 FAA) ethers (i.e. B72 and BS20), and oleyl (C18:1 FAA) ethers (i.e. BO20). The general
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compositions are shown in Tab. 1 and the basic structures are given in Fig. 1. All ethers can contain the free FAA and free PEGs.
Both lauryl ethoxylates B30 and Th can contain free C12 FAA which was not detected due to the volatility of the alcohol. B30 is C12 FAA etherified with an average of 4 EGU, while Th
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has a longer chain of an average of 9 EGU. In Fig. 4 both show a distribution pattern around
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49 – 52 min. Due to the polymeric macrogols used for etherification the resulting ethers show the same distribution which leads to the conclusion that this pattern must represent the
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higher chain length ethers. B30 showed a second distribution pattern near the theoretical
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retention range of C12 FAA (55 – 60 min) which might be ethers of shorter chain lengths and, thus, are more dominated by the lipophilic component. Both B30 and Th can contain free PEGs which are not clearly visible in Fig. 4, due to the scaling of the chromatographic
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overlay, but were found in the size range of approx. 8 – 16 EGU (reference PEG 400) for B30 and of approx. 10 – 35 EGU (reference PEG 600) for Th (for other scaling see
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“Supplemental material, Fig. B-1”).
Both cetyl ethoxylates B52 and BC10 can contain free C16 FAA which could be detected in
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the B52 sample. B52 is C16 FAA etherified with an average of 2 EGU which could be clearly identified in Fig. 4. In the time range of 70 – 75 min the free FAA eluted followed by the ethers. Due to the etherification with shorter PEGs the peak shape broadened but otherwise remained as plain single peaks not showing the typical polymeric distribution. With increasing
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average chain lengths such as in BC10 (average of 10 EGU) the polymeric distribution of PEGs dominates the ethers. Here, the ether distribution at 60 – 72 min covers the corresponding fatty alcohol. Both samples contain free PEGs which were found in the size range of approx. 10 – 35 EGU (reference PEG 600) for B52 and approx. 17 – 35 EGU (reference PEG 1000) for BC10 (for further information see “Supplemental material, Fig. B1”).
9
Stearyl ethoxylates B72 and BS20 can contain free C18 FAA which could be detected in B72, whereas in BS20 no free FAA was found. B72 is C18 FAA etherified with an average of 2 EGU which could be clearly identified in Fig. 4. In the time range of 88 – 95 min the free FAA eluted followed by the ethers. Due to the etherification with shorter PEGs the peak shape broadened but otherwise remained as single peaks not showing the typical polymeric distribution. With increasing average chain lengths such as in BS20 (average of 20 EGU) the polymeric distribution of PEGs dominates the ethers. Here, the ether distribution eluted in a time range of 70 – 90 min before the retention time of the corresponding FAA (95 min). Both
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samples could contain free PEGs but only in B72 free PEGs in the size range of approx. 10 – 40 EGU (references PEG 600 and PEG 1000) were found in low amounts at retention times of 15 – 20 min (for further information see “Supplemental material, Fig. B-1”).
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The oleyl ethoxylate BO20 consists of oleyl alcohol (C18:1 FAA) etherified with an average of 20 EGU. The substance can contain free oleyl alcohol and free PEGs. The sample is also
dominated by the polymeric distribution of the etherified PEGs. Three distribution patterns
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were found. The main pattern eluted in the time range of 60 – 70 min are the presumable C18:1 FAA ethers. The free FAA could not be detected due to the dominant ether distribution
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coeluting in the same time slot. The earlier eluting ether group (52 – 57 min) might be
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degraded ethers, whereas the later eluting ether group eluted in the time range of C18 FAA ethers (72 – 85 min) which might be possible by-products. Free PEGs were found in a wide
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size range of approx. 3 - > 50 EGU (references PEG 600 to PEG 1500) (for further information see “Supplemental material, Fig. B-1”).
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In general, a correlation between average number of EGU, etherified FAA and retention behavior can be drawn from the results shown in Fig. 4 for ethers of the same FAA and
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variable amount of EGU the retention time decreases with increasing chain length, i.e. the ethers of BS20 (20 EGU, stearyl alcohol) having shorter retention time ranges than the ethers of B72 (2 EGU, stearyl alcohol). Vice versa, ethers with an equal average number of
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EGU, such as for example B52 (2 EGU, cetyl alcohol) and B72 (2 EGU, stearyl alcohol) show a retention behavior depending on the etherified alcohol: macrogol ethers with
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C16 FAA (B52) had shorter retention times than macrogol ethers with C18 FAA (B72).
3.4.2. Macrogol fatty acid esters Macrogol stearates (M45, M52, M59) are esters of stearic acid and polyethylene glycol of a nominal average molecular weight. The substances consist of different ester types, depending on the type of stearic acid used for ethoxylation. The Ph. Eur. specifies two main types of stearic acid, i.e. type I, containing 40.0 – 60.0% C18 FA and, in sum, a minimum of 10
90.0% C16 FA plus C18 FA, as well as type II consisting of 90.0 – 99.0% of C18 FA and, in sum, a minimum of 96.0% C16 FA plus C18 FA [42]. Type II was used as a reference in this study. All macrogol stearates may contain the free corresponding FAs and free PEGs. M45 contains stearic esters with an average of 8 EGU. Since two polymeric distribution patterns are eluted in the time ranges of 59 – 63 min (C16 FA esters) and 70 – 77 min (C18 FA esters) separately, it seems likely that type I stearic acid was used for esterification. As shown in Fig. 5 the esters dominate the co-eluting corresponding free FAs. Free PEGs were found in a molecular sizes range of approx. 5 – 17 EGU (references PEG 300 and
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PEG 400).
M52 consists of stearic esters with an average of 40 EGU. The M52 sample also shows two
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polymeric distribution patterns which are eluted in the time ranges of 50 – 57 min (C16 FA esters) and 60 – 70 min (C18 FA esters) separately, so it is again likely that type I stearic acid was used for esterification. The polymeric ester distributions eluted significantly before the expected retention time of the corresponding FA. However, no free FAs were present in
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M52. Free PEGs were found in a molecular sizes range of approx. 17 – 54 (reference
N
PEG 1500).
A
M59 contains stearic macrogol esters of an average of 100 EGU. Due to the higher chain lengths and the wider size distribution the esters are highly dominated by the hydrophilic
M
polymeric macrogols which results in lower retention time ranges (approx. 25 – 38 min and 42 – 75 min). The free FAs C16 FA and C18 FA are clearly separated and detected. Free than PEG 3000).
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PEGs were located in a wide size range of approx. 8 - >100 (references PEG 1000 to more
PT
In RH40 the glycerol triesters of 12-hydroxystearic acid having an average of 40 EGU are more hydrophilic than the corresponding macrogol stearates due to an additional hydroxyl moiety. Additionally, steric effects, due to the branched triester structure (Fig. 6), might
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reduce interactions with the stationary phase. This resulted in much shorter retention times (18 – 23 min) of the polymeric ester distribution close to the free PEGs (Fig. 5). Free PEGs were found in a size range of approx. 13 – 30 EGU (references PEG 600 and PEG 1000). The prominent peak eluting at a retention time of about 45 min could not be identified. It
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might be the free 12-hydroxystearic acid or coeluting triglycerol esters, but this could not be reliably verified by LC-MS.
3.4.3. Polysorbates
11
Polysorbates consist of ethoxylated sorbic acid with an average of 20 EGU, one of the PEG side chains esterified with the corresponding FA which are C12 FA for T20 and C18:1 FA for T80, respectively. Both samples can contain free FAs and PEGs. In Fig. 7, the ester distribution (45 – 60 min) of T20 is shown which is dominated by the polymeric character of the corresponding PEGs. Due to the semi-volatility of C12 FA no free FA was detected. Free PEGs in the size range of approx. 10 – 40 EGU (references PEG 600 and PEG 1000) were found.
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T80 is characterized by an ester distribution (50 – 63 min) dominated by the polymeric character of the corresponding PEGs. Here, the free C18:1 FA was clearly separated from
the ester fraction and detected at a retention time of 65 min. Free PEGs were located in a
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size range of approx. 10 – 40 EGU (references PEG 600 and PEG 1000).
3.5. Summary of method characteristics
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3.5.1. Reproducibility
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For reproducibility triplicate measurements of all 0.1 mg/mL samples were performed. Each
A
injection of the triplicate measurements was executed on consecutive days to ensure inter-
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day reproducibility. The aim of reducing the sample concentration was to produce a comparable level of all samples and to examine recovery of each sample.
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All single reference substances (PEG, FA, FAA) were easily recovered and retention times were reproduced during the triplicate measurements; thus, the different substances could be distinguished and identified. The main distribution of the emulsifiers was recovered as well as
PT
the free PEGs which is shown for M45 as example substance in Fig. 8. Here, in all 4 samples, free PEGs are detected in a size range of PEG 300 to PEG 400 such as the
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polymeric ester distributions of C16 FA esters (59 – 63 min) and C18 FA esters (70 – 77 min). Both, the distribution of the macrogol polymers and the distribution of the esters could be reproduced.
As a negative result some of the possible by-products in macrogol stearate, such as esters of
A
other FA, e.g. C12 FA or C14 FA, were not detected within the test solutions (1) – (3), due to the lower concentration levels of 0.1 mg/mL and therefore, a lower sensitivity. In the reference solution (R; c = 1.0 mg/mL) these by-products were detected (Fig. 8). For the other emulsifiers studied, results were comparable.
12
3.5.2. Specificity Specificity was obtained for all reference substances (PEGs, FAs, and FAAs) which were clearly separated from each other, whereas some of the esters and ethers coeluted with the free precursor substances (e.g. BO20, M45). However, reducing the sample concentrations and, thus, reducing potential concentration-correlated peak-broadening effects, did not reduce the coelution. Hence, the specificity does not benefit from the reduction of sample
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concentration.
3.5.3. Sample preparation
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Considering the results of reproducibility (section 3.5.1) and specificity (section 3.1.2) sample concentrations are recommended as 0.1 mg/mL for references (PEGs, FAs, FAAs) and at least 1.0 mg/mL for emulsifiers to ensure the detectability of potential by-products and free
N
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precursor substances.
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4. Conclusion
The study describes a new HPLC-CAD method suitable for characterizing polymeric
M
excipients primarily based on macrogols. It can be considered as a supplement to the established methods described in the Ph. Eur. and allows reducing the analytical efforts.
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Additionally, the method provides a much better reproducibility and batch-to-batch monitoring, especially for the distribution of the oligomers and the detection of the individual components, than the determination of bulk parameters. Free macrogols could be completely
PT
separated into the single oligomers (n = 3 – 54). In some cases, free fatty acids and alcohols coeluted with the respective esters and ethers, whereas in other cases they were completely
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separated. Furthermore, the distribution of ester and ether oligomers could be evaluated for the first time.
Current protocols consider single components only, e.g. determining the amount of free fatty
A
acids and the fatty acid composition of polysorbates after saponification [19], the behavior of polysorbate under specific conditions, e.g. gastrointestinal hydrolysis [15] and oxidative degradation [18], and the distribution of polymeric macrogols [31]. The simultaneous determination of free macrogols, fatty alcohols and acids, and the respective ethers and esters using HPLC-CAD was not reported so far. In contrast to the established pharmacopoeial methods which are highly time-consuming and prone to errors, the described method is more comfortable and accurate. Whilst for adhering 13
to protocols such as determining the hydroxyl value the continuous presence of an analyst must be ensured, the HPLC-CAD method is able to run autonomously after sample preparation is done and thus can easily be automated. Furthermore, the method is compatible with LC-MS, as long as the respective molar masses are within the tolerable mass range of the instrumentation used. This enables identifying impurities which are produced during the production process or monitoring degradation
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products which is an important aspect during long term stability assessments.
Conflict of interest
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None of the authors stated a conflict of interest.
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Tables Table 1 Nominal composition of the analyzed macrogol-based emulsifiers and internal labeling. The internal labeling is used in Section 3. Only the main components are mentioned (EGU = ethylene glycol unit). Internal labeling1
Composition (suppliers’ information)/ further classification
Brij® 30
B30
average of 4 EGU etherified with lauryl alcohol [6]
Brij® 52
B52
average of 2 EGU etherified with cetyl alcohol [3]
Brij® 72
B72
average of 2 EGU etherified with stearyl alcohol [4]
Brij® C10
BC10
average of 10 EGU etherified with cetyl alcohol [2]
Brij® S20
BS20
average of 20 EGU etherified with stearyl alcohol [2]
Brij® O20
BO20
average of 20 EGU etherified with oleyl alcohol [2]
Polidocanol, Thesit®
Th
Cremophor® RH40
RH40
Polysorbate 80, Tween® 80
SC R
U
N
A
average of 40 EGU esterified with 12hydroxystearic acid and glycerol [2]
average of 40 EGU esterified with stearic acid [2]
M59
average of 100 EGU esterified with stearic acid [2]
T20
sorbitol anhydrides copolymerized with about 20 EGU, esterified with mainly lauric acid [2]
T80
sorbitol anhydrides copolymerized with about 20 EGU, esterified with mainly oleic acid [2]
PT
M52
A
Polysorbate 20, Tween® 20
average of 9 EGU etherified with lauryl alcohol [5]
average of 8 EGU esterified with stearic acid [2]
M45
CC E
Myrj® 59
M
ED
Myrj® 45 Myrj® 52
IP T
Substance
1
Internal labeling was assigned as follows: initial letter of the registered trade name (“B” for Brij®. “M” for Myrj®, “T” for Tween®) followed by the type number (i.e. Brij ® C10 as BC10). The two outstanding samples Thesit (“Th”) and Cremophor® RH40 (“RH40”) are labelled differently.
19
B [%]
0
10
1
10
6
15
7.5
15
17.5
30
27.5
32.5
35
32.5
45
75
50
80
70
85
105
85
A
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PT
ED
M
A
N
U
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t [min]
IP T
Table 2 Chromatographic conditions: time table of the linear gradient for the HPLCCAD method (flow rate = 1 mL/min; injection volume: 10 µL).
20
Table 3 Chromatographic conditions for LC-MS measurements. Different settings (1,2) for ion trap were used to receive different charging levels of the analytes. Flow rate
1.0 mL/min 1 µL 2 µL
PEG 300, PEG 400, PEG 600 PEG 1000, PEG 1500
Target masses
PEG 300 PEG 400 PEG 600 PEG 1000 PEG 1500
283; 415 371; 547 459; 679; 899 723; 899; 1119; 1339 1207; 1427; 1648; 1868
Setting (1)
stability nebulizer drying gas (nitrogen flow) drying temp.
20% 30.0 psi 8.00 L/min
stability nebulizer drying gas (nitrogen flow) drying temp.
100% 50.0 psi 10.00 L/min
PT
Gradient
SC R
U
N
A
350 °C
M
t [min] 0 1 6 7.5 17.5 27.5 35
350 °C
ED
Setting (2)
A
CC E
column re-equilibration: 10 min
21
IP T
Injection volume
B [%] 10 10 15 15 30 32.5 32.5
Figures
A
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A
N
U
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IP T
Fig. 1 General structures of macrogol-based emulsifiers and the analyzed precursor molecules (macrogols, FA and FAA). n/a: reference substances were not available.
22
IP T
Fig. 2 Separation of different PEGs using HPLC-CAD. Sample concentration for all: 0.1 mg/mL.
A
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PT
ED
M
A
N
U
SC R
Fig. 3 Separation of a mix containing 0.1 mg/mL of each fatty acid (FA: C12, C14, C16, C17, C18, C18:1, C18:2) and fatty alcohol (FAA: C16, C16/18).
23
SC R
IP T
Fig. 4 Separation results of alcohol ethoxylates. For PEG 3000 a sample concentration of 1 mg/mL sample was used. “Mix” = mixed sample of FA: C12, C16, C17, C18, C18:1, C18:2 and FAA: C16, C16/18.
A
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M
A
N
U
Fig. 5 Separation results of macrogol fatty acid esters. For PEG 3000 a sample concentration of 1 mg/mL sample was used. “Mix” = mixed sample of FA: C12, C16, C17, C18, C18:1, C18:2 and FAA: C16, C16/18.
24
IP T
Fig. 6 Structure of Cremophor® RH40.
A
CC E
PT
ED
M
A
N
U
SC R
Fig. 7 Separation results of polysorbates. For PEG 3000 a sample concentration of 1 mg/mL sample was used. “Mix” = mixed sample of FA: C12, C16, C17, C18, C18:1, C18:2 and FAA: C16, C16/18.
25
A
CC E
PT
ED
M
A
N
U
SC R
IP T
Fig. 8 Overlayed chromatograms of triplicate measurements (1) – (3) and reference runs (R) of M45 and the reference substances PEG 400 and C18 FA, respectively. Zoomed area of the relevant retention range (PEGs, FAs, FAAs, esters).
26