Method for Making Polyoxyethylene 1,4 Sorbitan Fatty Acid Ester

Abstract

The invention discloses a method for preparation of polyoxyethylene 1,4-sorbitan fatty acid ester, such as Polysorbate 80, by a reaction of polyoxyethylene 1,4-sorbitan with a fatty acid chloride, and polyoxyethylene 1,4-sorbitan fatty acid esters obtainable by this method.

Claims

1. A method for preparation of polyoxyethylene 1,4-sorbitan fatty acid ester by a reaction REAC-A of polyoxyethylene 1,4-sorbitan with an acid chloride ACIDCHLOR; ACIDCHLOR is compound of formula (I); ##STR00008## R1 is linear or branched C.sub.10-22 alkyl or linear or branched C.sub.10-22 alkenyl.

2. The method according to claim 1, wherein R1 is linear C.sub.10-22 alkyl or linear C.sub.10-22 alkenyl, the polyoxyethylene of the polyoxyethylene 1,4-sorbitan, has an average of from 10 to 30 ethylene oxide units, or a combination thereof.

3. (canceled)

4. The method according to claim 1, wherein REAC-A is done at a temperature TEMP-A, no solvent is used for REAC-A, no water is used for REAC-A, no catalyst is used for REAC-A, or a combination thereof, wherein TEMP-A is from 0 to 70° C.

5-7. (canceled)

8. The method according to claim 1, wherein REAC-A is done neat.

9. The method according to claim 1, wherein the polyoxyethylene 1,4-sorbitan is prepared by a reaction REAC-B, wherein 1,4-sorbitan is reacted with ethylene oxide.

10. The method according to claim 9, wherein the 1,4-sorbitan is prepared by a method SORBID comprising four consecutive steps STEP1, STEP2, STEP3 and STEP4, wherein in STEP1 D-sorbitol is dehydrated in a dehydration reaction DEHYDREAC in the presence of p-toluenesulfonic acid and tetrabutylammonium bromide, STEP1 provides a mixture MIX1; in STEP2 ethanol is mixed with MIX1, STEP2 provides a mixture MIX2; in STEP3 isopropanol is mixed with MIX2, STEP3 provides a mixture MIX3; in STEP4 1,4-sorbitan is isolated from MIX3.

11. The method according to claim 10, wherein the p-toluene sulfonic acid is used in form of p-toluenesulfonic acid monohydrate.

12. The method according to claim 10, wherein no solvent is used for DEHYDREAC, no water is charged for DEHYDREAC, DEHYDREAC is done neat, or a combination thereof.

13. (canceled)

14. (canceled)

15. The method according to claim 10, wherein STEP2 is done at a temperature TEMP2 of from 60 to 90° C. and/or STEP3 is done at a temperature TEMP3-1 of from 10 to 30° C.

16. (canceled)

17. The method according to claim 10, wherein after the mixing of isopropanol, STEP3 comprises a cooling COOL3 of MIX3 to a temperature TEMP3-2 of from −5 to 5° C., or STEP3 comprises stirring STIRR3 of MIX3, STIRR3 is done for a time TIME3-2, TIME3-2 is from 1 to 12 h.

18. (canceled)

19. The method according to claim 17, wherein STIRR3 is done after COOL3.

20. (canceled)

21. The method according to claim 10, wherein STEP1, STEP2 and STEP3 are done consecutively in one and the same reactor.

22. The method according to claim 9, wherein the 1,4-sorbitan is prepared by a method SORBIDAQU for preparation of 1,4-sorbitan with three consecutive steps STEP1AQU, STEP2AQU and STEP3AQU, wherein in STEP1AQU D-sorbitol is dehydrated in a dehydration reaction DEHYDREACAQU in the presence of p-toluenesulfonic acid and tetrabutylammonium bromide, STEP1AQU provides a mixture MIX1AQU; in STEP2AQU ethanol is mixed with MIX1AQU, STEP2AQU provides a mixture MIX2AQU; in STEP3AQU isopropanol is mixed with MIX2AQU, STEP3AQU provides a mixture MIX3 AQU; D-sorbitol is used for STEP1AQU in form of a mixture of D-sorbitol with water.

23. A polyoxyethylene 1,4-sorbitan fatty acid ester obtainable by the method for preparation of polyoxyethylene 1,4-sorbitan fatty acid ester by a reaction REAC-A, with the method and REAC-A as defined in claim 1.

24. A polyoxyethylene 1,4-sorbitan fatty acid ester according to claim 23, wherein the average number of ethylene oxide (EO) units of the PEO 1,4-sorbitan monoester species in said polyoxyethylene 1,4-sorbitan fatty acid ester is from 19 to 23, or the polyoxyethylene 1,4-sorbitan fatty acid ester contains 10 wt % or less of PEO isosorbide monooleate, the wt % based on the weight of the sample of the polyoxyethylene 1,4-sorbitan fatty acid ester which is analyzed for its content of PEO isosorbide monooleate.

25. A polyoxyethylene 1,4-sorbitan fatty acid ester which does not contain isosorbide species, sorbitol species, or both isosorbide species and sorbitol species.

26. (canceled)

27. A polyoxyethylene 1,4-sorbitan fatty acid ester which shows in a MALDI spectrum a signal distribution with only one maximum, or wherein the MALDI spectrum of said polyoxyethylene 1,4-sorbitan fatty acid ester shows no signals of substances with MW of over 3500 with signal heights of over 5% relative to the maximum of the whole distribution in the MALDI spectrum.

28. (canceled)

29. A polyoxyethylene 1,4-sorbitan fatty acid ester obtained by the method of claim 1 which does show: an endothermic signal in DSC with a maximum of the signal at a temperature of −13° C. or lower or an endothermic signal in DSC with a delta H of not more than 35 J/g.

30. (canceled)

31. A polyoxyethylene 1,4-sorbitan fatty acid ester obtained by the method of claim 1 which does not show: an endothermic signal in DSC with a maximum of the signal at a temperature of above −13° C., an endothermic signal is DSC with a delta H of more than 35 J/g, or an exothermic signal DSC with a maximum of the signal at a temperature of −50° C. or higher.

32-34. (canceled)

35. A method of forming a drug formulation comprising the polyoxyethylene 1,4-sorbitan fatty acid ester according to claim 23, as an excipient in the drug formulations.

36. (canceled)

Description

FIGURES

[0302] The descriptions in the figures means the following, if not otherwise stated:

TABLE-US-00001 DSC Exo{circumflex over ( )} Heat flow, exothermic heat flow is positive, endothermic heat flow is negative, if not otherwise stated MALDI intensity intensity in arbitrary units (a.u.) m/z mass divided by charge Preparative a.u. intensity in arbitrary units HPLC

[0303] FIG. 1 DSC measurement of Example 2, first heating cycle

[0304] FIG. 2 DSC measurement of Example 4, first heating cycle

[0305] FIG. 3 DSC measurement of Example 5, first heating cycle

[0306] FIG. 4 DSC measurement of Example 6, first heating cycle

[0307] FIG. 5 DSC measurement of Croda HP, first heating cycle

[0308] FIG. 6 DSC measurement of NOF, first heating cycle

[0309] FIG. 7 DSC measurement of Example 5, first (solid line) and second (dashed line) cooling cycle

[0310] FIG. 8 DSC measurement of Croda HP, first (solid line) and second (dashed line) cooling cycle

[0311] FIG. 9 DSC measurement of NOF, first (solid line) and second (dashed line) cooling cycle

[0312] FIG. 10 MALDI spectrum of Example 10

[0313] FIG. 11 MALDI spectrum of Example 2

[0314] FIG. 12 MALDI spectrum of Example 4

[0315] FIG. 13 MALDI spectrum of Example 5

[0316] FIG. 14 MALDI spectrum of Example 6

[0317] FIG. 15 MALDI spectrum of Example 10 overlaid with curve of Gaussian distribution function

[0318] FIG. 16 HPLC chromatogram of preparative HPLC of Example 5, overlay of UV absorption (solid line) and weight distribution (dashed line)

[0319] FIG. 17 HPLC chromatogram of preparative HPLC of Croda HP, overlay of UV absorption (solid line) and weight distribution (dashed line)

[0320] FIG. 18 HPLC UV chromatogram of preparative HPLC, overlay of Example 5 (solid line) and Croda HP (dashed line)

[0321] FIG. 19 HPLC weight chromatogram of preparative HPLC, overlay of Example 5 (solid line) and Croda HP (dashed line)

[0322] FIG. 20 Illustration of the analysis of the area of the endothermic valley in the DSC measurement of Example 5, first (solid line) and second (dashed line) heating cycle, the scaling of the y-axis is still normalized indicating the dimension, just without giving the location of the 0 W/g.

[0323] FIG. 21 HPCL UV chromatogram of preparative HPLC of Croda HP

[0324] FIG. 22a MALDI spectra: Comparison of (A) Example 5, (B) NOF, (C) Croda HP (FIG. 22a) and (D) Croda SR (FIG. 22b)

[0325] FIG. 22b MALDI spectra: Comparison of (A) Example 5, (B) NOF, (C) Croda HP (FIG. 22a) and (D) Croda SR (FIG. 22b)

[0326] FIG. 23 Polysorbate Synthesis of Croda

[0327] FIG. 24 Raw Materials for the two product ranges of Polysorbate of Croda

[0328] FIG. 25 Process differences of Croda HP and Croda SR

[0329] FIG. 26 Color difference of Croda HP and Croda SR

[0330] FIG. 27 MALDI spectrum of the Croda SR

[0331] FIG. 28 DSC measurement of Croda SR, first heating cycle

[0332] FIG. 29 DSC measurement of Croda SR, first (solid line) and second (dashed line) cooling cycle

[0333] FIG. 30a Gaussian distribution function is fitted to the left side of the mass distribution of (A) Example 5, (B) NOF (FIG. 30a), (C) Croda HP and (D) Croda SR (FIG. 30b)

[0334] FIG. 30b Gaussian distribution function is fitted to the left side of the mass distribution of (A) Example 5, (B) NOF (FIG. 30a), (C) Croda HP and (D) Croda SR (FIG. 30b)

[0335] FIG. 31a Three Gaussian curves fitted to each the three peaks of the MALDI spectra of (B) NOF (FIG. 31a), (C) Croda HP and (D) Croda SR (FIG. 31b), as well as the one Gaussian curve fitted to the one peak in the MALDI spectrum of Example 5 (FIG. 31a).

[0336] FIG. 31b Three Gaussian curves fitted to each the three peaks of the MALDI spectra of (B) NOF (FIG. 31a), (C) Croda HP and (D) Croda SR (FIG. 31b), as well as the one Gaussian curve fitted to the one peak in the MALDI spectrum of Example 5 (FIG. 31a).

[0337] FIG. 32a One Gaussian curve fitted to all signals of the MALDI spectra of (B) NOF (FIG. 32a), (C) Croda HP and (D) Croda SR (FIG. 32b), as well as the one Gaussian curve fitted to all signals in the MALDI spectrum of Example 5 (FIG. 32a).

[0338] FIG. 32b One Gaussian curve fitted to all signals of the MALDI spectra of (B) NOF (FIG. 32a), (C) Croda HP and (D) Croda SR (FIG. 32b), as well as the one Gaussian curve fitted to all signals in the MALDI spectrum of Example 5 (FIG. 32a).

[0339] FIG. 33 Calibration curves prepared with the three solutions of PEO isosorbide oleate (concentrations 0.001, 0.002 and 0.006 mg/ml)

EXAMPLES

Materials

[0340] The following materials were, if not stated otherwise:

TABLE-US-00002 No Quality Chemicals Sources (Batch/Source) (wt %) PEO sorbitan Example 10 Oleic acid Green Oleo Srl 6936 91.6 Cremona, Italy Thionyl chloride Acros Organics 169490010 99.5+ Oxalyl chloride Acros Organics 129610010 98 Oleoyl chloride Sigma Aldrich 367850 >89

[0341] Density of thionyl chloride: 1.683 kg/L

[0342] NOF Polysorbate 80 (HX2)™, Lot 704352, NOF Corporation, Tokyo, Japan [0343] With MALDI Isosorbide species, such as PEO-isosorbide or PEO-isosorbide-fatty acid ester, and also sorbitol species, such as sorbitol ester ethoxylates, are detectable. [0344] (H).sup.13C NMR method: Isosorbide species, such as PEO-isosorbide or PEO-isosorbide-fatty acid ester, are detectable. [0345] (A) HPLC-ELSD method: Isosorbide species, such as PEO-isosorbide or PEO-isosorbide-fatty acid ester, are detectable. [0346] The MALDI spectrum shows a signal distribution with three maxima. [0347] Croda HP Tween® 80HP-LQ-(MH), also called Tween 80 HP, “HP” means “High Purity”, batch number 0001176143, Chemical Description: Polysorbate 80, Croda Europe Limited, 62920 Chocques, France [0348] With MALDI Isosorbide species, such as PEO-isosorbide or PEO-isosorbide-fatty acid ester, and also sorbitol species, such as sorbitol ester ethoxylates, are detectable. [0349] (H).sup.13C NMR method: Isosorbide species, such as PEO-isosorbide or PEO-isosorbide-fatty acid ester, are detectable. [0350] (A) HPLC-ELSD method: Isosorbide species, such as PEO-isosorbide or PEO-isosorbide-fatty acid ester, are detectable. [0351] The MALDI spectrum shows a signal distribution with three maxima. [0352] Croda SR SUPER REFINED® POLYSORBATE 80-LQ-(MH), batch number 0001186606, Chemical Description: Polysorbate 80, Croda Europe Limited, Cowick Hall, Snaith, Goole, DN14 9AA, East Riding of Yorkshire, GB [0353] With MALDI Isosorbide species, such as PEO-isosorbide or PEO-isosorbide-fatty acid ester, and also sorbitol species, such as sorbitol ester ethoxylates, are detectable. [0354] (H).sup.13C NMR method: Isosorbide species, such as PEO-isosorbide or PEO-isosorbide-fatty acid ester, are detectable. [0355] (A) HPLC-ELSD method: Isosorbide species, such as PEO-isosorbide or PEO-isosorbide-fatty acid ester, are detectable. [0356] The MALDI spectrum shows a signal distribution with three maxima.

TABLE-US-00003 D-Sorbitol 98 wt % TsOH−H.sub.2O 99 wt % TBAB 98 wt % Ethanol 99 wt % Isopropanol 99 wt %

Methods:

(A) HPLC-ELSD

[0357] HPLC-ELSD is a reversed phase HPLC using Evaporative Light Scattering Detection.

[0358] Column: Agilent Zorbax Eclipse XDB-C18 (150 mm×3 mm; 3.5 micrometer)

[0359] Pump: [0360] min pressure: 5 bar [0361] may pressure: 400 bar [0362] max flow gradient: 100 mL/min.sup.2 [0363] Eluent A: ultra pure H.sub.2O [0364] Eluent B: isopropanol [0365] Gradient:

TABLE-US-00004 Time Flow [min] [ml/min] % A % B 0.0 0.3 98 2 1 0.3 98 2 19 0.3 80 20 64 0.3 0 100 71 0.3 0 100 73 0.3 98 2 83 0.3 98 2

[0366] Injection: [0367] Injection volume 10 microlitre

[0368] Autoinjektor: [0369] Syringe Volume 100 microliter [0370] Injection Mode Injection with needle wash/washing solution: Acetonitrile

[0371] Detector [0372] Detector Type ELSD [0373] Temperature 60° C. [0374] Pressure (Gas) 3.5 bar [0375] Gain 10 [0376] Filter 8 s

[0377] Column oven [0378] Temperature 20° C.

[0379] SAT/IN [0380] Unit mV [0381] Description ELSD [0382] Scale Factor 1000 [0383] Sampling rate 10

[0384] Typical Integration Parameters [0385] Peak Width 250 [0386] Threshold 20 [0387] Inhibit Integration 42-56 min

[0388] Sample preparation:

[0389] 50 mg+/−5 mg sample were dissolved in 50 ml of acetonitrile.

[0390] The percentage determined by an HPLC chromatogram are the area percentage of the respective signal.

[0391] The LOD (Limit of Detection) with a Signal-to-noise ratio of 3 was 0.06 area-%.

[0392] The LOQ (Limit of Quantification) with a Signal-to-noise ratio of 10 was 0.20 area-%. [0393] No signals with an area-% in HPLC chromatogram of 0.06 or greater means that no isosorbide is detectable. [0394] Signals with an area-% in HPLC chromatogram of from 0.06 to 0.20 means that isosorbide is detectable but not yet quantifiable. [0395] Signals with an area-% in HPLC chromatogram of 0.20 or greater means isosorbide is quantifiable.

(B) DSC

[0396] All measurements were measured in an identical way, the samples were used as such, if not otherwise stated, with sample weights ranging from 2 to 12 mg for the different products. If not otherwise stated the samples were dried in a vacuum pistol over night at room temperature, then they were immediately sealed in a glove bag into 40 microliter aluminum pans with pins, Mettler Toledo, in order to avoid and minimize any uptake of humidity from the atmosphere, and then the pans were subjected to DCS measurements with a DSC 1 STARe system from Mettler Toledo. The samples were run from 25 to −80° C., equilibrated for 5 min at −80° C., then heated from −80 to +80° C., equilibrated for 5 min at +80° C. (denoted 1st cycle). Then this thermal cycle was repeated, +80 to −80° C., equilibration at −80° C., −80 to +80° C., equilibration at +80° C. and then back to 25° C., with all heating and cooling segments at 10° C./min. If not stated otherwise, the heating segments from the first thermal cycle are displayed. If nothing else is reported, the measurement of the second heating cycle produced the same signal as the measurement of the first heating cycle, thereby it was confirmed that the samples did not show any thermal history.

(C) MALDI and DSC from a Preparative HPLC and from Non-Fractionated Samples

(C1) Sample Preparation and Preparative HPLC

[0397] All samples were used as such, if not otherwise stated. The samples were dissolved in ACN to provide a solution with a concentration of 300 mg/ml. 300 microliter of this solution were injected (Waters sample manager 2700) and loaded onto a C18 column (Xterra Prep MS C18 OBD, 5 micrometer, 19×100 mm, Waters). The polysorbate species were separated using an ACN:H2O gradient starting at 45% ACN and increasing to 100% in 30 min with a flow rate at 10 ml/min and a column temperature of 50° C. (Thermostated column compartment TCC-100, Dionex). The separation continued at 100% ACN until reaching 120 min and no more species could be detected. The species were detected with a UV detector (Waters 2487 dual absorbance detector) at 195 nm (lamda max with epsilon=11000 for C═C bonds present in oleic acid). The MassLynx V4.0 software was used for data acquisition. 10 ml fractions were manually collected in 20 ml glass tubes. From each tube 10 microliter were taken out for MALDI analysis prior to evaporation until dryness under vacuum (GeneVac centrifugal evaporator EZ-2, SP Scientific). The evaporated fractions were then used for DSC analysis.

(C2) MALDI of Samples from Preparative HPLC (C1) and of Non-Fractionated Samples

[0398] 2.5-Dihydroxybenzoic acid (super-DHB>=99.0%, Sigma Aldrich) was used as matrix and prepared as a 5 mg/ml solution in EtOH with 10 mM NaCl added in order to exclusively detect sodiated adducts. Prior to use, the matrix was sonicated for 10 min in a bath in order to obtain a solution. Non-fractionated samples were dissolved in ethanol to provide a solution with a concentration of 5 mg/ml, and for the HPLC fractions the 10 microliter samples were used without further preparation. All samples were mixed 1:1 (vol:vol) with the matrix and vortexed before spotting 1 microliter of each sample onto a target plate (MPT 384 polished steel, Bruker) in triplicates. All sample spots were allowed to dry and crystallize on the plate before MALDI measurements were performed. Positive ion MALDI-TOF mass spectrometry was carried out on an Ultraflex TOF/TOF, Bruker Daltonics instrument equipped with a 337 nm N.sub.2 laser operated at a frequency of 5 Hz in reflection mode. Spectra were recorded at an accelerating voltage of 25 kV and with matrix suppression until 450 Da with 1000 summed acquisitions per measurement. The laser power was kept slightly above the threshold for detection (usually ca. 40%) in order to get optimal peak resolution. All mass spectra were acquired with FlexControl 3.4 and analyzed with the FlexAnalysis 3.4 software.

(C3) DSC of Samples from Preparative HPLC (C1)

[0399] The evaporated samples from the preparative HPLC separation were extracted from the 20 ml tubes by dissolving in acetone and transfer (with three washes) to 1.5 ml glass vials equipped with 0.1 ml micro-inserts (Sigma Aldrich). The samples were then evaporated to dryness under vacuum (GeneVac centrifugal evaporator EZ-2, SP Scientific). All samples were afterwards dried overnight in a vacuum pistol before they were transferred to DSC pans (40 microliter, aluminum pans with pins, Mettler Toledo) and sealed in a vacuum bag at controlled humidity (ca. 7% or lower) to avoid uptake of moisture from the atmosphere. The DSC measurements were done as described under the method description (B) DCS

(D) GC (1,4-Sorbitan)

Instrument Parameters

[0400]

TABLE-US-00005 Colum DB-1 HT (30 m * 0.25 mm * 0.1 μm) Agilent Technologies, Santa Clara, USA Temperature program: Initial; time 100° C.; 0 min Rate1; Final 1; Time 1 8° C./min; 350° C.; keep 10 min Run Time 41.25 min Equilibration Time 0.5 min Mode Cons. flow Carrier gas H.sub.2 Flow 1.5 ml/min Split ratio 10:1 Inlet Temperature 350° C. Injection Volume 1 microliter Detector temperature 350° C.

Sample Preparation

Sample Stock Solution

[0401] Add 2 g sample to 5 ml pyridine and 10 ml acetic anhydride in a screw-cap bottle (25 mL) and heat up to 120° C. for 2 hours under stirring.

Sample Solution

[0402] 0.5 ml of Sample stock solution is added into an auto sampler vial with 1 ml of dichloromethane and mixed

[0403] 1,4-Sorbitan is detected at ca. 12.3 min.

(E).SUP.1.H NMR

[0404] .sup.1H NMR is a routine analytical method for the skilled person, so only one exemplary set of parameters is given in the following which can be used:

[0405] Solvent: DMSO-d6

[0406] 5 to 10 mg of sample are dissolved in 0.6 ml of DMSO-d6 and mixed.

(F).SUP.13.C NMR

[0407] .sup.13C NMR is a routine analytical method for the skilled person, so only one exemplary set of parameters is given in the following which can be used:

[0408] Solvent: DMSO-d6

[0409] 20 to 50 mg of sample are dissolved in 0.6 ml of DMSO-d6 and mixed well.

(G) Optical Rotation Method (1,4-Sorbitan)

Instrument Parameters

[0410]

TABLE-US-00006 Instrument MCP 300 of Anton Paar GmbH, Graz, Austria Wavelength 589 nm Cell 100.00 mm Temperature 20.0° C. Response 2 s Measure N = 5 Delay 10 s Stable Temperature ±0.3° C.

Sample Preparation

Blank

[0411] Pure water

Sample Solution

[0412] 300±3 mg of 1,4-Sorbitan was added into a 100 ml volumetric flask, then dissolved with water and diluted to volume.

(H).SUP.13.C NMR Method for Verifying if Isosorbide Species are Present of not

[0413] The samples were dissolved in deuterated chloroform prior to the measurements.

[0414] Approximately 90 to 120 mg of material were mixed with 0.55 ml of d1-chloroform. 0.5 ml of solution was filled in 5 mm NMR tube. The .sup.1H-decoupled .sup.13C-NMR, .sup.13C(.sup.1H)-NMR, were performed with proton decoupling and nuclear Overhauser effect (NOE). The measurements were carried out at 25° C., on a 400 MHz spectrometer at a resonance frequency of 100.61 MHz. The samples were run with 8192 scans using a pulse length of 14.5 micro-sec (90°), a 20 Hz spin, an acquisition time of 1.301 s, and a relaxation delay of 5 s. 32768 complex data points were collected, using a spectral width of 25188.9 Hz (250 ppm). All spectra were Fourier transformed with a line broadening of 1 Hz and zero filling to 128 k data points. The spectra were phase and baseline corrected, and the chloroform peak was used as a reference peak, determined to 77.23 ppm relative to TMS for the .sup.13C-NMR.

Example 1—Oleoyl Chloride with Thionyl Chloride

[0415] A two-neck round bottom flask equipped with a stir bar was charged with oleic acid (12.62 g, 40.9 mmol, 1.0 equiv) and the flask was purged with N.sub.2. After heating to 40° C., thionyl chloride (12.5 ml, 172.0 mmol, 4.2 equiv) was added dropwise over 10 min by an addition funnel while stirring, gas evolution was observed. Then the temperature was increased to 65° C. and the reaction mixture was stirred for 1 hour. Then the reaction mixture was cooled to room temperature. Excess SOCl.sub.2 was removed by a rotary evaporator followed by drying under vacuum providing oleoyl chloride. The yield of oleoyl chloride was assumed to be 100%.

Example 2—Polysorbate 80 with 1.0 Equiv Oleoyl Chloride from Thionyl Chloride

[0416] PEO sorbitan (47.1 g, 42.9 mmol, 1.0 equiv), prepared according to Example 10, were weighed into a single-neck round bottom flask and the atmosphere in the flask was exchanged for N.sub.2. Oleoyl chloride, the whole amount that was prepared according to Example 1, was added at room temperature and the reaction mixture was stirred for 15 min at room temperature.

[0417] The mixture steam distilled under reduced pressure of ca. 80 mbar for ca. 10 min. The pH was raised by this steam distillation from ca. 1.5 to ca. 4.5.

[0418] The product from the steam distillation was used as is for analysis. [0419] (H).sup.13C NMR method: No isosorbide species, such as PEO-isosorbide or PEO-isosorbide-fatty acid ester, were detectable. [0420] (A) HPLC-ELSD method: No isosorbide species, such as PEO-isosorbide or PEO-isosorbide-fatty acid ester, were detectable.

Example 3—Oleoyl Chloride with Thionyl Chloride

[0421] A two-neck round bottom flask equipped with a stir bar was charged with oleic acid (30.0 g, 97.3 mmol, 1.0 equiv) and the flask was purged with N.sub.2. After heating to 40° C., thionyl chloride (30 ml, 413.0 mmol, 4.2 equiv) was added dropwise over 10 min by an addition funnel while stirring, gas evolution was observed. Then the temperature was increased to 65° C. and the reaction mixture was stirred for 1 hour. Then the reaction mixture was cooled to room temperature. The excess SOCl.sub.2 was removed by a rotary evaporator followed by drying under vacuum providing oleoyl chloride. The yield of oleoyl chloride was assumed to be 100%.

Example 4—Polysorbate 80 with 1.2 Equiv Oleoyl Chloride from Thionyl Chloride

[0422] PEO sorbitan (22.4 g, 21.4 mmol, 1.0 equiv), prepared according to Example 10, were weighed into a single-neck round bottom flask and the atmosphere in the flask was exchanged for N.sub.2. Oleoyl chloride (7.74 g, 25.7 mmol, 1.2 equiv, prepared according to example 3) was added at room temperature and the reaction mixture was stirred for 15 min at room temperature.

[0423] The mixture steam distilled under reduced pressure of ca. 80 mbar for ca. 10 min. The pH was raised by this steam distillation from ca. 1.5 to ca. 4.5.

[0424] The product from the steam distillation was used as is for analysis. [0425] (H).sup.13C NMR method: No isosorbide species, such as PEO-isosorbide or PEO-isosorbide-fatty acid ester, were detectable. [0426] (A) HPLC-ELSD method: No isosorbide species, such as PEO-isosorbide or PEO-isosorbide-fatty acid ester, were detectable.

Example 5—Polysorbate 80 with 1.4 Equiv Oleoyl Chloride from Thionyl Chloride

[0427] Example 4 was repeated with the difference that 1.4 equiv oleoyl chloride were added instead of 1.2 equiv. [0428] (H).sup.13C NMR method: No isosorbide species, such as PEO-isosorbide or PEO-isosorbide-fatty acid ester, were detectable. [0429] (A) HPLC-ELSD method: No isosorbide species, such as PEO-isosorbide or PEO-isosorbide-fatty acid ester, were detectable.

Example 6—Polysorbate 80 with 1.6 Equiv Oleoyl Chloride from Thionyl Chloride

[0430] Example 4 was repeated with the difference that 1.6 equiv oleoyl chloride were added instead of 1.2 equiv. [0431] (H).sup.13C NMR method: No isosorbide species, such as PEO-isosorbide or PEO-isosorbide-fatty acid ester, were detectable. [0432] (A) HPLC-ELSD method: No isosorbide species, such as PEO-isosorbide or PEO-isosorbide-fatty acid ester, were detectable.

Example 7—Oleoyl Chloride with Oxalyl Chloride

[0433] A two-neck round bottom flask equipped with a stir bar was charged with oleic acid (2.0 g, 7.1 mmol, 1.0 equiv) and the flask was purged with N.sub.2. DCM (6.5 mL) was added, a clear solution formed. Then oxalyl chloride (1.21 ml, 14.2 mmol, 2.0 equiv) was added dropwise at room temperature over 10 min by an addition funnel while stirring, then the reaction mixture was stirred at room temperature for 2 hour. The DCM and excess oxalyl chloride were removed at the rotary evaporator followed by drying under vacuum. The yield of oleoyl chloride was assumed to be 100%.

Example 8—Polysorbate 80 with 1.0 Equiv Oleoyl Chloride from Oxalyl Chloride

[0434] PEO sorbitan (7.4 g, 7.1 mmol, 1.0 equiv), prepared according to Example 10, were weighed into a single-neck round bottom flask and the atmosphere in the flask was exchanged for N.sub.2. Oleoyl chloride, the whole amount that was prepared according to example 7, was added at room temperature and the reaction mixture was stirred for 15 min at room temperature. The product was used as is for analysis. [0435] (H).sup.13C NMR method: No isosorbide species, such as PEO-isosorbide or PEO-isosorbide-fatty acid ester, were detectable. [0436] (A) HPLC-ELSD method: No isosorbide species, such as PEO-isosorbide or PEO-isosorbide-fatty acid ester, were detectable.

Example 9—Polysorbate 80 with 1.0 Equiv Commercially Available Oleoyl Chloride

[0437] PEO sorbitan (5.96 g, 5.7 mmol, 1.0 equiv), prepared according to Example 10, were weighed into a single-neck round bottom flask and the atmosphere in the flask was exchanged for N.sub.2. Oleoyl chloride (1.885 mL, 5.7 mmol, 1.0 equiv, Sigma Aldrich) was added at room temperature and it was stirred for 15 min at this temperature.

[0438] The product was used as is for analysis. [0439] (H).sup.13C NMR method: No isosorbide species, such as PEO-isosorbide or PEO-isosorbide-fatty acid ester, were detectable. [0440] (A) HPLC-ELSD method: No isosorbide species, such as PEO-isosorbide or PEO-isosorbide-fatty acid ester, were detectable.
Results from (A) HPLC-ELSD

[0441] Table 1 shows the HPLC-ELSD results of Examples 1, 4, 5, 6, 8 and 9, reported are the area % of the elution peaks of the Mono-, Di- and Tri-ester (denoted with “Mono”, “Di” and “Tri” in the Table 1) in the respective HPLC chromatogram; the first value is the absolute percentage of the area of the respective peak (“abs %”) based on the total peak area of the chromatogram, the second value is the percentage of the area of the respective peak based on the sum of the areas of the three peaks (“rel”).

[0442] The maximum of the elution peak is observed: [0443] between 27.4 and 27.7 min for the Mono-ester [0444] between 40.3 and 40.6 min for the Di-ester [0445] between 47.2 and 47.3 min for the Tri-ester

[0446] The elution peaks of the three esters are well separated from each other.

TABLE-US-00007 TABLE 1 Mono Di Tri Ex abs % rel % abs % rel % abs % rel % 2 28.1 60.2 13.8 29.6 4.73 10.2 4 30.3 56.0 17.5 32.3 6.3 11.7 5 30.1 43.2 27.3 39.1 12.3 17.7 6 27.3 33.8 34.4 42.5 19.3 23.7 8 29.5 61.0 14.8 30.5 4.13 8.5 9 28.9 62.2 13.8 29.7 3.8 8.1
Results from (B) DSC

[0447] Table 2 shows the DSC results, values of T(peak) and for delta H are an average of 3 DCS analysis per sample in case of Croda HP and NOF, whereas they are values of one DSC analysis in case of Example 2, 4, 5 and 6.

TABLE-US-00008 TABLE 2 T(peak) delta H Ex FIGURE [° C.] [J/g] Cycle Endothermic Peaks 2 FIG. 1 −31.5 0.13 Heating 1st cycle 4 FIG. 2 −39.5 0.32 Heating 1st cycle 5 FIG. 3 −40.6 0.42 Heating 1st cycle 6 FIG. 4 −39.3 0.49 Heating 1st cycle Croda HP FIG. 5 −11.6 47.7 Heating 1st cycle NOF FIG. 6 −6.5 42.0 Heating 1st cycle Croda SR FIG. 28 −7.4 46.3 Heating 1st cycle Exothermic Peaks Croda HP FIG. 8 −35.2 41.7 Cooling 1st Cycle Croda HP FIG. 8 −35.4 41.4 Cooling 2nd Cycle NOF FIG. 6 −46.1 36.4 Heating 1st cycle Croda SR FIG. 29 −41.5 32.4 Cooling 1st and 2nd Cycle

Discussion of the Curves of the Heating Cycles:

[0448] The Croda HP shows in the heating cycle a distinct endothermic peak, which is interpreted to be a melting peak, with a delta H of ca. 48 J/g, at ca. −12° C. (FIG. 5) [0449] The NOF shows in the heating cycle two distinct peaks: [0450] an endothermic peak, which is interpreted to be a melting peak, with a delta H of ca. 42 J/g, at ca. −7° C.; [0451] an exothermic peak, which is interpreted to be a melting peak, with a delta H of ca. 36 J/g, at ca. −46° C. (FIG. 6). [0452] The Croda SR shows in the heating cycle distinct endothermic peak, which is interpreted to be a melting peak, with a delta H of ca. 46.3 J/g, at ca. −7.4° C. (FIG. 28) [0453] The DSC of the four Examples 2, 4, 5 and 6 show a slight, non-distinct, not well defined and rather broad endothermic valley between ca. −30 to −40° C. with a delta H of from 0.1 to 0.5 J/g (FIGS. 1 to 4), which is smaller by ca. a factor 100 compared to the delta H of the melting peaks of Croda HP and NOF. The determination of the area of this slight endothermic valley by the program of the DSC instrument is demonstrated in FIG. 20. [0454] In the curves there appears an exothermic hump directly before, that is still at lower temperature, said slight endothermic valley (FIGS. 1 to 4), which cannot be clearly interpreted, since it may well be just a irregularity in the baseline due to the rather fast heating rate of 10° C. per min and due to its very small size. Its area is only ⅓ of the area of the already slight endothermic valley, making it even less significant.

[0455] The DSC of Example 8, 9 and 13 look similar to the DSC of the four Examples 2, 4, 5 and 6. So Examples 2, 4, 5, 6, 8, 9 and 13 do not show at all or at least not clearly a melting of crystallization behavior.

Discussion of the Curves of the Cooling Cycles:

[0456] Croda HP shows in the first cooling cycle a distinct exothermic peak with a delta H of ca. 42 J/g at ca. −35° C.; in the second cooling cycle there a distinct exothermic peak with a delta H of ca. 41 at ca. −35° C. which shows a distinct shoulder at ca. −30° C.; due to its shoulder it has a shape distinctly different from the peak in the first cooling cycle (FIG. 8). [0457] Croda SR shows in the first and second cooling cycle the more or less same distinct exothermic peak with a delta H of ca. 32.4 J/g at ca. −41.5° C. (FIG. 29).

[0458] Neither NOF nor Examples 2, 4, 5, 6, 8, 9 and 13 show a peak in any of the two cooling cycles (FIG. 7 (illustrative for the Examples 2, 4, 5, 6, 8, 9 and 13) and FIG. 9).

Results from (C) MALDI and DSC from a Preparative HPLC

[0459] The samples have been tested by MALDI. Example 5 and Croda HP were examined in detail by separation on a preparative HPLC and fractionation into 100 individual fractions, which were consecutively collected between 0 and 100 min, so each fraction was collected for 1 min (10 ml fractions), and that were analyzed by MALDI. The actual weight of all fractions was determined and a weight distribution was created and overlaid with the UV chromatogram: [0460] FIGS. 16 and 17: HPLC chromatogram of preparative HPLC with overlay of UV absorption (solid line) and weight distribution (dashed line), Example 5 and Croda HP respectively [0461] FIGS. 18 and 19: HPLC chromatogram of preparative HPLC, overlay of Example 5 (solid line) and Croda HP (dashed line), UV absorption and weight distribution respectively

[0462] From this separation pure fractions of PEO sorbitan mono oleate were tested by DSC: Fractionation of Example 5 yielded pure PEO sorbitan monoester fractions, which also did not show any melting peaks in DSC analysis.

[0463] In a MALDI mass spectrum all ethoxylated distributions are separated by 44 Da, which is equal to one EO unit. In order to calculate the average EO content of a mass distribution the mass peak list was exported and fitted to a Gaussian distribution function:

[00001]$f\left(x\right)={\mathrm{ae}}^{-\frac{{\left(x-b\right)}^2}{2c^2}}$

[0464] Where a is the height of the Gaussian distribution function, b is the position of the Gaussian distribution function center and c can be used as an estimate of the EO spread or dispersity of the Gaussian distribution function around the center mass. The Gaussian distribution function center position thus indicates the mass of the molecule present in the mixture, which gives the highest MALDI peak.

[0465] In the case of Example 10, the PEO sorbitan, this value corresponded to 1146 Da. A sodiated PEO sorbitan with 21 EO units has a molecular mass of 1112 Da and a sodiated PEO sorbitan with 22 EO units has a mass of 1156 Da. The average integer EO number for this Gaussian distribution function will thus be estimated to be 22 (FIGS. 10 and 15: Example 10 without and with overlay of the curve of the Gaussian distribution function).

[0466] The same methodology can be used for analysis of the pure PEO sorbitan monooleate fractions. Non-fractionated products, though, contain PEO sorbitan mono- di and tri-esters with overlapping distributions due to the fact that one oleate, having 264 Da, is isobaric with six EO units. The mass peak of a sodiated PEO sorbitan monooleate with 20 EO units has 1332 Da and therefore falls on top of a PEO sorbitan diester with 14 EO units and a PEO sorbitan triester with 8 EO units. It is therefore not possible to calculate the average EO content from a MALDI mass spectrum of non-fractionated samples alone, even with the knowledge of the weights of the HPLC fractions. However, with the knowledge gained from fractionated samples, the average EO content of the non-fractionated samples can be estimated.

Isosorbide Based Species and PEO Esters:

[0467] FIG. 18 shows the overlay of Example 5 (solid line) and Croda HP (dashed line) of the UV absorption. The UV absorption shows between ca. 16 and 27 min major signals. In general, the HPLC column in connection with the gradient that was used (from polar to non-polar) separates according to polarity, the higher polar species elute earlier then the less polar species, so the monoester species elute first, then the diester and later on the species with more than two ester residues. Using MALDI based on the preparative HPLC samples, the monoester species were further analyzed for the distribution of their molecular weights. In the same way, the major signals between ca. 30 and 46 min have been identified and assigned to diester species.

[0468] In case of the monoester species, which elute between ca. 16 and 27 min: [0469] the major signals of Example 5 have been assigned to PEO sorbitan monoester species with varying number of EO units; [0470] the major signals of Croda HP have been assigned to [0471] PEO sorbitan monoester species with varying number of EO units, to [0472] PEO isosorbide monoester species with varying number of EO units, and to [0473] PEO monoester, that is to polyoxyethylated fatty acid esters, with varying number of EO units.

[0474] In case of the diester species, which elute between ca. 30 and 46 min: [0475] the major signals of Example 5 have been assigned to PEO sorbitan diester species with varying number of EO units; [0476] the major signals of Croda HP have been assigned to [0477] PEO sorbitan diester species with varying number of EO units, and to [0478] PEO isosorbide diester species with varying number of EO units. [0479] PEO diester, that is to PEG with fatty acid esters on both sides, with varying number of EO units.

[0480] The isosorbide based species and the PEO ester species elute noticeably later than the sorbitan species, and this both in case of the mono- and of the diester species, even though there is an overlap due to the distribution of the molecular weight which is caused by the distribution of the number of EO units.

[0481] The isosorbide based species and the PEO ester species actually more or less coelute. FIG. 21 illustrates the time ranges where the various species elute in case of Croda HP [0482] A: Peak of PEO sorbitan monoester species [0483] B: Peak of PEO isosorbide monoester and PEO monoester species [0484] C: Peak of PEO sorbitan diester species [0485] D: Peak of PEO isosorbide diester and PEO diester species

[0486] Also in case of Example 5 also PEO esters were observed, but only in trace or small amounts in comparison to the major peaks of the PEO sorbitan esters.

[0487] In case of Example 10 also PEO isosorbides were observed, but only in trace amounts just above the noise level in comparison to the major peaks of the PEO sorbitan.

[0488] Isosorbide species have not been observed in Example 5.

Estimation of the Average Number of EO Units of the PEO 1,4-Sorbitan Monoester Species in Example 5, NOF, Croda HP and Croda SR:

[0489] In general any of the ethoxylated species in Example 5 shows lower number of EO units in comparison the respective species in Croda HP and in Croda SR, the difference is always roughly between 5 and 10 EO units. FIG. 22a and FIG. 22b illustrate how this shift of the average number of EO units affects the m/z distribution of the MALDI spectrum: [0490] A: Example 5 [0491] B: NOF [0492] C: Croda HP [0493] D: Croda SR

[0494] Obviously the MALDI peaks in case of Example 5 have been shifted to lower m/z values compared to NOF, Croda HP and Croda SR.

[0495] The MALDI mass distribution of pure 1,4-sorbitan monoester fractions fits well to a Gaussian distribution function. In the case of non-fractionated material, that is Example 5, NOF, Croda HP and Croda SR, the main mass distribution contains overlapping mass distributions due to the presence of PEO sorbitan mono- di- and tri-oleate, which are all isobaric molecules. The polyester species are present in a lower amount than the monoesters but will shift the total mass spectrum slightly towards higher masses. A MALDI mass distribution from a non-fractionated sample will thus deviate from a Gaussian distribution function. If, however, a Gaussian distribution function is fitted to the left side of the mass distribution as illustrated in FIG. 30a and FIG. 30b, the center mass peak (b, the position of the Gaussian distribution function center) is a good estimation of the average number of EO units of the 1,4-sorbitan monoester species. This estimation was tested and verified for three samples (Example 5, NOF, Croda HP and Croda SR) of which the two samples Example 5 and Croda HP had been subjected to fractionation and detailed analysis, results are given in Table 3:

TABLE-US-00009 TABLE 3 Sample b (m/z) Average EO units (A) Example 5 1331 20 (B) NOF 1660 27 (C) Croda HP 1659 27 (D) Croda SR 1745 29

MALDI of Examples 2, 4, 5, 6, 8, 9, 13 Shows Absence of Isosorbide Species or of Sorbitol Species:

[0496] With MALDI no isosorbide species, such as PEO-isosorbide or PEO-isosorbide-fatty acid ester, were detectable in the Examples 2, 4, 5, 6, 8, 9 and 13. [0497] With MALDI no sorbitol species, such as sorbitol ester ethoxylates, were detectable in the Examples 2, 4, 5, 6, 8, 9 and 13.

MALDI of Example 5, NOF, Croda HP and Croda SR for Analysis of Width of Distribution and of Number of Maxima:

[0498] The MALDI of Example 5 shows a distribution of signals with only one maximum, whereas the MALDI of NOF, Croda HP and Crode SR show in the signal distribution in addition to a main maximum two additional maxima; one of the additional maxima has a b value at a lower m/z value relative to the b value of the main maximum, the other additional maximum has a b value at a higher m/z value relative to the b value of the main maximum. Both additional maxima have a lower intensity than the main maximum.

[0499] Table 4 shows the b values of the three Gaussian curves fitted to each the respective maximum, as well as the b value of the Gaussian curve fitted to the one maximum in the MALDI spectrum of Example 5. These fitted curves are illustrated in FIG. 31a and FIG. 31b.

TABLE-US-00010 TABLE 4 b (m/z) fit of the fit of the fit of the Sample left maximum main maximum right maximum (A) Example 5 — 1392 — (B) NOF 900 1774 2788 (C) Croda HP 886 1727 2553 (D) Croda SR 930 1795 2797

[0500] The MALDI spectrum of Examples 2, 4, 5, 6, 8, 9 and 13 show a signal distribution with only one maximum.

[0501] This difference of the products according to instant invention versus the known polysorbates products can also be illustrated when only one Gaussian curve is fitted to all the signals, that is to the whole distribution, in a MALDI spectrum. The c value of the Gaussian distribution function can be used as an estimate of the spread of the m/z values of the signals, that is of the dispersity of the Gaussian distribution function around the center m/z value of the Gaussian distribution function, which is expressed by the b value. Table 5 shows these c values for Example 5, NOF, Croda HP and Croda SR.

[0502] This fit of one Gaussian curve to all the signals in the MALDI spectrum is illustrated in FIG. 32a and FIG. 32b.

TABLE-US-00011 TABLE 5 c (m/z) one fit of the Sample whole distribution (A) Example 5 440 (B) NOF 816 (C) Croda HP 785 (D) Croda SR 981

Example 10—PEO Sorbitan from 1,4-Sorbitan Using 20 EO

[0503] 200 g Naphtha (petroleum), heavy alkylate, CAS 64741-65-7, 100 g (0.61 mol, 1 equiv) 1,4-sorbitan, prepared according to Example 11, and 0.6 g KOH were charged into a 4 L autoclave. The autoclave was rendered inert by evacuating first and then applying afterwards 0.5 bar pressure with N.sub.2, this was done for four times in total.

[0504] The mixture was heated to 150° C., 553 g (12.6 mol, 20.7 equiv) ethylene oxide were added in such speed that the temperature did not raise above 160° C. and the pressure did not raise above 3.8 bar; the addition was done in 4 h. Then the mixture was stirred for 2 h at 150° C.

[0505] After cooling to 60° C. 2.3 g AcOH were added. Two phases formed, one with solvent, the other with product, and were separated. Residual solvent was removed by steam distillation at a rotary evacuator. 625 g product was obtained.

[0506] Yield: 95% based on the assumption that a PEO sorbitan with an average of 20 EO was obtained. This assumption was also applied when this product was used in further reactions. .sup.1H-NMR and .sup.13C-NMR confirmed the structure.

[0507] DSC analysis showed no sign of crystallization or melting, neither in both heating cycles nor in both cooling cycles.

Example 11—1,4-Sorbitan

[0508] D-sorbitol (300 g, 1.647 mol, 1 equiv) was charged into a 1.5 L reactor. p-Toluenesulfonic acid monohydrate (2.665 g, 0.014 mol, 0.0085 (0.85%) equiv) was charged, followed by charging of TBAB (9.6 g, 0.03 mol, 0.0182 (1.81%) equiv). Vacuum of reactor 4 to 6 mbar was applied. Then the mixture was heated to 110° C. (the mixture melted at around 90° C.) and stirred at 110° C. for 6 hours. The mixture was cooled to 70 to 75° C. in 30 min. Ethanol (150 mL) was charged. The resulting mixture was stirred at 70 to 75° C. for 2 hours and formed a clear solution. Then the solution was cooled to 20° C. in 3 hours. A yellow suspension was formed. Isopropanol (150 mL) was charged. The mixture was cooled to 0° C. in 1 hour. The mixture was slurry at 0° C. for 4 hours. The mixture was filtered, and the cake was washed with isopropanol (150 mL). The cake was dried at 50° C. for 16 hours under vacuum to provide 142.2 g of product as white solid.

[0509] Yield 52.6%

[0510] .sup.1H NMR and .sup.13C NMR confirmed the structure.

[0511] GC area-%: [0512] 1,4-Sorbitan 97% [0513] Isosorbide 0.14% [0514] D-Sorbitol 0.12%

[0515] Specific Rotation: −22.26°, c=3.1 (water)

Comparative Example 1

[0516] From Nov. 4 to 7, 2018, on the Walter E Washington Convention Center, Washington, D.C., the conference “aaps 2018 PharmSci 360” was held with a Move-In on Friday, Nov. 2, 2018 and Pre-Conference Activities on Saturday, Nov. 3, 2018.

[0517] From 9:00 am to 5:00 pm of these Pre-Conference Activities Workshops and Short Courses took place. One of these Workshops took place between 9.45 AM and 10:15 AM with the title: “SC1-Synthesis and Control of Polysobates for Bioüharmaceuticel Applications”, which was held by Sreejit R. Menon, representing the company CRODA, www.crodahealthcare.com, Croda, Inc., Edison, N.J., USA.

[0518] The presentation showed on slide 12 the Polysorbate Synthesis of Croda, see FIG. 23, which uses the sequence:

[0519] Sorbitol-(Dehydration)->Sorbitan-(Esterification with Fatty Acid)->Sorbitan Fatty Ester-(Ethoxylation)->Polysorbate-(Finishing)->High quality Polysorbate.

[0520] According to this sequence Crode produces two product ranges: [0521] Croda HP, also called Tween 80 HP, the abbreviation “HP” means “high purity” [0522] Croda SR, the abbreviation “SR” means “super refined”, also called “SR PS 80” (meaning super refined polysorbate 80), Super Refined Polysorbate”

[0523] Slide 11, see FIG. 24, lists the Raw Materials for these two product ranges.

[0524] On Slide 17, see FIG. 25, the differences of Croda HP and Croda SR: [0525] 1. the process differences for the SR grade versus the HP grade are: [0526] Higher purity starting materials (fatty acid & sorbitol) [0527] Manufactured under milder conditions, preventing carmelization [0528] Process controlled during every step [0529] 2. color difference: the HP grade has a yellowish color, whereas the SR grade is almost colorless, this is illustrated on Slide 16, see FIG. 26. The original presentation was not black-white, but colored, but still the gray scale reproduction shows the yellowish color of Croda HP in form of a darker hue compared to the sample of Croda SR.

[0530] In Slide 15, see FIG. 27, the MALDI spectrum of the SR grade is shown. Three dominant peaks areas are characterized by the chemical species which give rise to these peak areas: [0531] 1. Isosorbide ester Ethoxylates & PEG [0532] 2. Sorbitan ester ethoxylates [0533] 3. Sorbitol ester ethoxylates

[0534] Clearly the MALDI spectrum shows even for the SR grade, which is the grade with the highest purity that is currently available on the market not only the one desired peak area of Sorbitan ester ethoxylates, but also significant peak areas caused by the presence of isosorbide and sorbitol derivatives, which are present in the SR grade.

Example 12—Oleoyl Chloride

[0535] A two-neck round bottom flask was charged with oleic acid (469.3 g, 1.64 mol, 1.0 equiv) and the flask was purged with N.sub.2. Dichloromethane (DCM) (1520 mL) was added, a clear, colorless solution formed. Then oxalyl chloride (288 ml, 3.3 mol, 2.0 equiv) was added dropwise at room temperature over 50 min while stirring, then the reaction mixture was stirred at room temperature for 2 h. The DCM and excess oxalyl chloride were removed at the rotary evaporator at ca. 35° C. and ca. 450 to 8 mbar followed by drying under vacuum. The yield of oleoyl chloride was assumed to be 100%.

Example 13—Polysorbate 80

[0536] PEO sorbitan (1001.9 g, 0.96 mol, 1.0 equiv, prepared according to Example 10) were weighed into a 21 reactor and the atmosphere in the flask was exchanged for N.sub.2. Oleoyl chloride (435.5 g, 1.34 mol, 1.4 equiv, prepared according to Example 12) was added at room temperature during ca. 40 min and the reaction mixture was stirred for 1 h at room temperature. Then the reaction mixture was heat up to 60° C. and vacuum was applied under stirring (200 mbar) for 1 day.

[0537] The formed HCl could be removed and the pH increased to 5.9. The pH was measured preparing a solution of a sample of the product in water with a content of 5 wt % of the sample. [0538] (H).sup.13C NMR method: No isosorbide species, such as PEO-isosorbide or PEO-isosorbide-fatty acid ester, were detectable. [0539] (A) HPLC-ELSD method: No isosorbide species, such as PEO-isosorbide or PEO-isosorbide-fatty acid ester, were detectable.

Example 14—Quantification of PEO Isosorbide Monooleate

Preparation of Calibration Material

[0540] The prep-HPLC method as described under (C1) Sample Preparation and preparative HPLC (except for the last sentence “The evaporated fractions were then used for DSC analysis.”) was used to separate PEO isosorbide oleate, prepared according to example 15. The material eluted, in time similar to the second peak, the peak B (see FIG. 21 as example) in the separation process of PS80. Three clean fractions were extracted and combined to get a broad PEO distribution and a large amount of material. The material was identified as PEO isosorbide monooleate using MALDI (method as described under (C2) for the HPLC fractions)

[0541] PEO isosorbide oleate with an average of 12 EO units, which is needed for standardization purpose, can be synthesized according to known procedures, in this example the PEO isosorbide oleate prepared according to example 15, was used.

LC-MS(ESI)

[0542] The isosorbide calibration material, the PEO isosorbide oleate, prepared according to example 15, was dissolved into three separate solutions: at 0.001 mg/ml, 0.002 mg/ml, 0.006 mg/ml. 10 microliter of each of the three solutions was injected into the LC (Water 2795 Alliance HT, Waters AG, 5405 Baden-Dättwil, Switzerland) and loaded onto a C18 column (Luna C18(2), 3 micrometer, 75×4.6 mm, Phenomenex, 63741 Aschaffenburg, Germany). The analyte species were separated using an can (Acetonitrile): H.sub.2O gradient starting at 45 vol % of ACN and increasing to 100 vol % of ACN in 45 min with a flow rate at 0.8 ml/min and a column temperature of 50° C. The separation continued at 100% ACN until reaching 60 min. The species were detected with a mass spectrometer (Waters Micromass Quattro Micro™) equipped with an electrospray ionization source (ESI). The MassLynx V4.0 software was used for data acquisition. Full scan mass spectra were acquired between m/z 200 and 2000 at a speed of 1 scan per second. The parameters for the MS scans were as follows: a desolvation gas temperature of 300° C., ion source temperature of 100° C., a nitrogen gas flow rate of 500 L/hour, nebulizing (N.sub.2) gas pressure was 6 bar, capillary voltage was 3000 V, and the cone voltage was 30 V.

Calibration Curve for PEO Isosorbide Monooleate

[0543] Mass spectra were collected and combined over the peak of interest using the MassLynx V4.0 software. The mass spectra were combined, ranging from the time when PEO isosorbide monooleate species were detected, (elution times between 28 to 34 min depending on sample). Each calibration concentration corresponds to one mass spectrum, used for the calibration curve. Four different distributions were detected in each spectrum, corresponding to four different adducts: Na+, K+, H+ and H.sub.2O. Each adduct distribution displayed a range of peaks, separated by 44 Da, corresponding to one EO unit. The intensity of all peaks of each distribution was added together, given four intensities, one for each adduct (see figure, circle: sum of all adducts, square: H.sub.2O adduct, triangle: H+ adduct, star: Na+ adduct, diamond (visible in the FIG. 33 in the vicinity of the triangles): K+ adduct) and calibration concentration. A calibration curve was calculated (using a standard linear regression) for each adduct, see FIG. 1, over the 0.001 to 0.006 mg/ml range. FIG. 33 shows the curves.

[0544] Two calibration curves were used, one for the H.sub.2O adduct (dashed line) and one for the K+ adduct (continuous line), to determine to PEO isosorbide content as these peaks do not overlap with PEO monooleates in the polysorbate samples.

Determination of Amount of PEO Isosorbide Monooleate in Polysorbate 80 Products

[0545] Two polysorbate samples, Croda HP and a polysorbate prepared according to Example 13, were dissolved in H.sub.2O to provide a solution with concentration of 0.05 mg/ml. One combined mass spectrum for each sample was collected, using the same method as for the isosorbide calibration material, the PEO isosorbide oleate, for the peak eluting between 28 to 34 min (sample dependent). The intensities for each adduct distribution was calculated, and the calibration curves were used to calculate the amount of PEO isosorbide monooleate species (in wt % based on the weight of the sample) for each sample. The polysorbate prepared according to Example 17 contained 1 wt % PEO isosorbide monooleate. The Croda HP contained more than 12 wt % PEO isosorbide monooleate, a specific concentration could not be determined as it was outside the scope of the calibration range.

[0546] The wt % are based on the weight of the respective polysorbate sample, the Croda HP and the polysorbate prepared according to Example 17.

Detection Limit:

[0547] The saturation of the detector occurs with 10 microliter of a PEO isosorbide oleate solution with a concentration above 0.006 mg/ml, to be more specific, between 0.006 mg/ml and 0.01 mg/ml is injected, this is equal to an amount of between 0.06 microgram and 0.1 microgram of PEO isosorbide oleate. Since 10 microliters of sample solutions of a concentration of 0.05 mg/ml are injected, this injection is equal to an amount of 0.5 microgram of sample material injected. Therefore the detection limit is between 12 wt % and 20 wt %.

Example 15—PEO Isosorbide Monooleate

[0548] Oleic acid (204.1 g) and DCM (660 ml) were mixed, oxalyl chloride (185 g) were added at 20° C. during 40 min, after stirring for 2 h at 20° C. the reaction mixture was concentrated at 33° C. from 450 to 22 mbar, obtained was a yellow, clear liquid (216.6 g).

[0549] PEO isosorbide (254.5 g, prepared according to example 16) were weighed into a 21 reactor and the atmosphere in the flask was exchanged for N.sub.2. Oleoyl chloride (160.9 g of the 216.6 g) was added at room temperature during 30 min and the reaction mixture was stirred for 40 min at room temperature. Then the reaction mixture was heat to 60° C. and vacuum was applied under stirring (200 mbar) for 1.5 day.

[0550] The formed HCl could be removed and the pH increased to 3.8. The pH was measured preparing a solution of a sample of the product in water with a content of 5 wt % of the sample.

Example 16—PEO Isosorbide from 1,4-Sorbitan Using 12 EO

[0551] 200 g Naphtha (petroleum), heavy alkylate, CAS 64741-65-7, 89.1 g (0.61 mol, 1 equiv) isosorbide (Sigma-Aldrich), and 0.6 g KOH were charged into a 4 L autoclave. The autoclave was rendered inert by evacuating first and then applying afterwards 0.5 bar pressure with N.sub.2, this was done for four times in total.

[0552] The mixture was heated to 150° C. 333 g (7.6 mol, 12.4 equiv.) ethylene oxide were added in such speed that the temperature did not raise above 160° C. and the pressure did not raise above 3.8 bar; the addition was done in 4 h. Then the mixture was stirred for 2 h at 150° C.

[0553] After cooling to 60° C. 1.4 g AcOH were added. Two phases formed, one with solvent, the other with product, and were separated. Residual solvent was removed by steam distillation at a rotary evacuator. ca. 376 g product was obtained.

[0554] Yield: 95% based on the assumption that a PEO sorbitan with an average of 120 EO was obtained. This assumption was also applied when this product was used in further reactions.

[0555] .sup.1H-NMR and .sup.13C-NMR confirmed the structure.

[0556] DSC analysis showed no sign of crystallization or melting, neither in both heating cycles nor in both cooling cycles.

Example 17—Polysorbate 80 with 22 EO

[0557] PEO sorbitan (502, 0.44 mol, 1.0 equiv, prepared according to Example 18) were weighed into a 21 reactor and the atmosphere in the flask was exchanged for N.sub.2. Oleoyl chloride (215.8 g, 0.7 mol, 1.5 equiv, prepared according to Example 12) was added at room temperature during ca. 40 min and the reaction mixture was stirred for 1 h at room temperature. Then the reaction mixture was heat up to 60° C. and vacuum was applied under stirring (200 mbar) for 3 days.

[0558] The formed HCl could be removed and the pH increased to 6.9. The pH was measured preparing a solution of a sample of the product in water with a content of 5 wt % of the sample.

Example 18—PEO Sorbitan from 1,4-Sorbitan Using 22 EO

[0559] 200 g Naphtha (petroleum), heavy alkylate, CAS 64741-65-7, 100 g (0.61 mol, 1 equiv) 1,4-sorbitan, prepared according to Example 11, and 0.6 g KOH were charged into a 4 L autoclave. The autoclave was rendered inert by evacuating first and then applying afterwards 0.5 bar pressure with N.sub.2, this was done for four times in total.

[0560] The mixture was heated to 150° C. 612 g (13.92.6 mol, 22.8 equiv) ethylene oxide were added in such speed the temperature did not raise above 160° C. and the pressure did not raise above 3.8 bar; the addition was done in 4 h. Then the mixture was stirred for 2 h at 150° C.

[0561] After cooling to 60° C. 2.5 g AcOH were added. Two phases formed, one with solvent, the other with product, and were separated. Residual solvent was removed by steam distillation at a rotary evacuator. 688 g product was obtained.

[0562] Yield: 95% based on the assumption that a PEO sorbitan with an average of 22 EO was obtained. This assumption was also applied when this product was used in further reactions.

[0563] .sup.1H-NMR and .sup.13C-NMR confirmed the structure.

[0564] DSC analysis showed no sign of crystallization or melting, neither in both heating cycles nor in both cooling cycles.