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Journal of Chromatographic Science , 2017, 1 – 8 doi: 10.1093/chromsci/bmx036 Article

Journal of Chromatographic Science, 2017, 1 8

doi: 10.1093/chromsci/bmx036

Article

Journal of Chromatographic Science , 2017, 1 – 8 doi: 10.1093/chromsci/bmx036 Article

Article

Esteri cation of Ibuprofen in Soft Gelatin Capsules Formulations Identi cation, Synthesis and Liquid Chromatography Separation of the Degradation Products

Michal Dou ša 1, * , Ludk Meca 1 , Petr Gibala 1 , Josef Jirman 1 , Marcela Tkadlecová 1 , Jan Srbek 1 , Jana Š alandová 1 , Eva Koval íková 2 , and Ji í B ichá1

1 ZentivaA SanoCompany, U Kabelovny 130, 102 37 Prague 10, Czech Republic and 2 Saneca Pharmaceuticals, Nitrianska 100, 920 27 Hlohovec, Slovak Republic

* Author to whom correspondence should be addressed. Zentiva, k.s. A Sano Company, U Kabelovny 130, 102 37 Prague 10, Czech Republic. Email: michal.dousa@seznam.cz

Received 29 April 2016; Revised 4 January 2017; Editorial Decision 31 March 2017

Abstract

Unknown impurities were identied in ibuprofen (IBU) soft gelatin capsules (SGCs) during long- term stability testing by a UHPLC method with UV detection and its chemical formula was deter- mined using high resolution/accurate mass (HRAM) LCMS. Reference standards of the impurities were subsequently synthesized, isolated by semi-preparative HPLC and characterized using HRAM LCMS, NMR and IR. Two impurities were formed by esterication of IBU with polyethylene glycol (PEG), which is used as a ll of the SGCs, and were identied as IBUPEG monoester and IBUPEG diester. Two other degradants arised from reaction of IBU with sorbitol and sorbitan, which are components of the shell and serves as plasticizers. Thus, IBU sorbitol monoester (IBUsorbitol) and IBU sorbitan monoester (IBUsorbitan ester) were identied. An UHPLC method was further opti- mized in order to separate, selectively detect and quantify the degradation products in IBU SGCs.

Introduction

Ibuprofen (IBU), 2-(4-isobutylphenyl) propanoic acid, is well-known non-steroidal anti-inammatory drug (NSAID) that inhibits prosta- glandin synthesis and has anti-pyretic and non-narcotic analgesic properties. Recently, the world production of IBU is in the vicinity of 15,000 tons per year ( 1). It is widely used orally in tablet, capsule or soft gelatin capsule (SGC) form ( 2 ). Generally, SGC formulation is gradually being more preferred for increased rate of drug absorp- tion, improved bioavailability of poorly soluble compounds, due to the technological reasons and for consumer preference (3 ). SGCs consist of a liquid ll enveloped by a one-piece hermetically sealed elastic outer shell. The formulation of the hydrophilic ll is typically based on polyethylene glycols (PEGs). The shell of a SGC is com- posed of gelatin, a plasticizer and water. Only a few plasticizers are

currently in use, namely glycerol, sorbitol/sorbitan mixtures and propylene glycol ( 4). The chemical stability of an active pharmaceutical ingredient (API) in pharmaceutical preparations is dependent on the formula- tion composition, technology and storage environment such as tem- perature, humidity and light. The potential physical and chemical interactions between drugs and excipients can affect the chemical nature, the stability and bioavailability of drugs and, consequently, their therapeutic efcacy and safety (5 10). The API can also react with trace contaminants that the excipients introduce, resulting in degradation (11 ). Stability testing provides information about how the quality of drug product varies with time under the inuence of various environmental factors and helps to determine recommended storage conditions and establish retest periods and shelf lives (12 ).

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Douš a et al.

Several HPLC methods for IBU determination and its related com- pounds and impurities in pharmaceutical preparations have been described in the literature. The most common technique for IBU quantication is RP-HPLC using an octadecylsilane column as sta- tionary phase and UV detection ( 1316 ). Capillary electrochromato- graphy using RP-18 packed capillary was used for simultaneous separation of IBU and its impurities (Ph. Eur. impurity A, B, C, E) as well ( 17, 18). Although degradation chemistry of IBU API and IBU in tablet formulations was well described (1822), only limited data related to stability of IBU in SGCs formulation has been reported (23). Forced degradation of IBU was observed in tablets containing PEG and polysorbate 80. Unfortunately, the structures of IBU degrada- tion products were not determined. Moreover, only accelerated deg- radation but not long-term stability data were reported (24). Decrease of esterication reaction between IBU and PEG was described when PEG in combination with polyvinylpyrrolidone was used as a SGCs ll ( 25). Formation of IBU PEG ester and its hydro- lysis was described in EMEA withdrawal assessment report for com- bination product IBU/diphenhydramine hydrochloride (26 ). Besides that, IBU PEG esters were prepared and their potential as a drug prolonged release system was investigated. However, these studies were aimed solely to preparation of new IBU polymeric prodrugs and studying of the IBU degradation was not the concern ( 27, 28 ). Further, the various types of poly(ethylene glycol) (PEG)IBU conju- gates by the nucleophilic substitution of bromo-terminated PEG with IBU-Cs salt were synthesized (29 ). This article describes identi cation of degradation products which were found in IBU SGCs pharmaceutical preparations using UHPLC method with UV detection. The impurities were synthesized, puri ed by semi-preparative HPLC and character- ized using HRAM LC MS, NMR and IR analysis. All character- ized degradants are esters formed b y drug-excipient interactions. Two degradants were formed by esteri cation of IBU with ll of

the SGCs and were identi ed as

IBU PEG monoester and IBU

PEG diester. Two other impurities arised from reaction of IBU with content of the shell and to best of our knowledge has not been reported yet. These unknow n degradants were determined as IBU sorbitol monoester (IBU sorbitol) and IBU sorbitan mono-

ester (IBU sorbitan).

sorbitol) and IBU sorbitan mono- ester (IBU – sorbitan). Figure 1. Long-term stability of IBU SGCs

Figure 1. Long-term stability of IBU SGCs in climatic chamber at 25 °C and 60% relative humidity. Content of impurities IBU PEG monoester (circles), IBUsorbitol (squares) and IBUsorbitan (triangles) as average from three batches is depicted. Content of IBUPEG diester was bellow reporting thresh- old and therefore was not monitored during stability testing.

Experimental

Reagents and chemicals

Acetonitrile HPLC gradient grade (J.T. Baker, USA), methanol (J.T. Baker, USA) and water puried by Milli-Q system (Millipore, USA) were used for preparation of samples, reference solutions and mobile phases. All other chemicals ( ortho-phosphoric acid, formic acid, dichlormethane, dicyclohexylcarbodiimide and 4-dimethylami- nopyridine) were of analytical grade or pure grade quality (Sigma- Aldrich, Czech Republic). The standard of IBU (in-house standard, purity 99.8%) was prepared in Zentiva (Czech Republic). PEG 600 (range of average molecular weight: 570630, dynamic viscosity at 20° C of 50% solution: 18 mPa s, hydroxyl value: 188 mg KOH/g) was obtained from Clariant Produkte (Germany). Polysorb 85 con- tained 43% of D-sorbitol and 27% of 1,4-sorbitan and was obtained from Roquette (France). The shell of in-house prepared SGCs com- posed of gelatin, partially dehydrated sorbitol liquid, carmine red and puried water in mass ratio 44:22:0.05:34. The ll of the SGCs composed of IBU, PEG 600, potassium hydroxide 85% and water in mass ratio 58:29:8:5.

HPLC instrumentation and methods

All chromatographic experiments were carried out on an Acquity UPLC system with a photodiode array detector (Waters, USA). The system control, data acquisition and processing was accomplished by the Empower software (Waters, USA). Chromatographic separa- tions were performed on an Acquity BEH C18 column (100 × 2.1 mm 2 , 1.7 μm; Waters, Czech Republic). The mobile phase was pumped at 0.5 ml/min, the injection volume was 1 μl and analytes were monitored at a wavelength of 220 nm. The gradient elution employed solutions A and B as mobile phase components. Method I: The solvent A was 0.1% ortho-phosphoric acid, while solvent B

solvent A was 0.1% ortho -phosphoric acid, while solvent B Figure 2. Structures of IBU –

Figure 2. Structures of IBUPEG monoester (A), IBU PEG diester (B), IBU sorbitol (C) and IBUsorbitan (D).

Esterication of Ibuprofen in Soft Gelatin Capsules FormulationsIdentication, Synthesis and Liquid

3

Formulations — Identi fi cation, Synthesis and Liquid 3 Figure 3. Preparation of IBU – PEG

Figure 3. Preparation of IBUPEG esters (A), IBUsorbitol (B) and IBU sorbitan (C).

was acetonitrile. The gradient program was set as follows: time/% of solvent B: 0/10, 2.0/10, 17/80, 20/80, 20.5/10 with an equilibra- tion time of 2 min. The column was thermostated at a temperature of 35° C. Method II : The solvent A was 0.1% ortho-phosphoric acid and solvent B was acetonitrile. The gradient program was set as fol- lows: time/% of solvent B: 0/5, 60/90, 61/5 with an equilibration time of 2 min. The column was thermostated at a temperature of 45° C. Method III : The solvent A was water and solvent B was meth- anol. The gradient program was set as follows: time/% of solvent B:

0/0, 70/85, 90/85, 91/0 with an equilibration time of 4 min. The col- umn was thermostated at a temperature of 65° C.

LCMS instrumentation and methods

High resolution/accurate mass (HRAM) MS experiments were per- formed on a LTQ XL Orbitrap Mass Spectrometer (Thermo, San

Jose, USA) coupled to an HPLC HTS PAL system (CTC Analytics, Switzerland). LC separation was performed on a Kinetex C18, 150 × 4.6 mm 2 , 5 μm (Phenomenex, Torrance, USA) column using 1.0 ml/min ow rate. The gradient elution employed solutions A and B as mobile phase components. The solvent A was 0.1% formic acid, while sol- vent B was acetonitrile. The gradient program was set as follows:

time/% of solvent B: 0/43, 17/43, 25/100, 30/100, with an equilibra- tion time of 7 min. For an ionization of the analytes an APCI ion source was operated in the positive ion mode (desolvation tempera- ture 400°C, capillary temperature 300°C, discharge current 4 μA and tube lens voltage 40 V).

Semi-preparative instrumentation and methods

Preparative HPLC separation and fraction collection was carried out on a Waters Auto purication system (System uidics organizer,

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Douš a et al.

Table I. NMR Assignments for IBU Esters

Position

δ(C)

δ(H)

Mult.

Integ. H

J (H,H) Position

δ (C)

δ (H)

Mult.

Integ. H

J (H,H) δ (C)

δ (H) Mult.

Integ. H J (H,H)

IBU PEG monoester

 

IBU sorbitol

IBU sorbitan 139.7 –– 129.0 7.10 d 127.1 7.20 d 137.9 –– 44.2 2.41 d 29.6 1.80 Nonet 22.2 0.85 d 44.2 3.75 q 18.8 1.39 d 174.0 –– 67.4 4.31 m

 

1 = 2

22.3

0.86

d

6

6.7

1

139.7

––

3

30.1

1.81

Nonet

1

~7

2 = 6 3 = 5

129.0

7.10

d

2

8.0

2

8.0

4

44.91

2.41

d

2

7.3

127.1

7.20

Overlap

2

2

8.0

5

140.4

––

4

138.0

––

6

129.2

7.05

d

2

8.2

7

44.2

2.41

d

2

7.1

2

7.2

7

127.1

7.18

d

2

8.2

8

29.6

1.80

Nonet

1

6.6

1

6.8

8

137.5

––

9 = 12

22.2

0.85

d

6

6.6

6

6.6

9

44.87

3.69

Overlap 1

10

44.2

3.74

Overlap

1

1

7.1

10

18.5

1.46

d

3

7.2

11

18.7

1.38

Overlap

3

3

7.1

11

174.6

––

13

174.1

––

12, 13

72.54

3.58

16

67.1

3.8

4.3

4 dd (overlap) Approx. 2

1

70.49 68.94 3.51 3.64

Overlap Approx. total intensity 52

 

66.9

 

66.3

 

63.78

4.19

66.2

3.85 m

1

61.56

3.69

14

Not observed ––

 

17,19,21, 23

68 74 (13 signals) 3.3 3.8

Overlap

Overlap

80.4

3.66

dt

1

8.3, 2.9

 

76.3

3.95 Overlap 1

75.4

3.91 Overlap 1

65.9

 

18,20,22,24,26

18,22,24

4.3

––

5.0

Broad signals Overlap

3.83 Overlap 1

5.04 br.s

––

 

4.90

 

4.83

 

25

63.4

3.3 3.6 Overlap

Overlap

73.5 3.89 m

1

 

62.4

3.44 m

1

Note : δ in ppm, J in Hz. Reference for IBU PEG monoester 1 H and 13 C was CDCl 3 signal. Reference for IBU sorbitol and IBU sorbitan was DMSO signal: 1 H δ (DMSO) = 2.5 ppm and 13 C δ (DMSO) = 39.50 ppm.

Esterication of Ibuprofen in Soft Gelatin Capsules FormulationsIdentication, Synthesis and Liquid

5

Table II. MS Accurate Mass Elemental Composition of IBU Esters

Impurity

Measured mass (m/z )

Theoretical mass (m/z )

Mass error

Molecular formula [M + H] + Fragments/adducts mass (m/z )

Fragments/adducts formulas

(ppm)

IBU PEG monoester

515.3224

515.3215

1.75

C 27 H 47 O 9 , n = 7 C 39 H 71 O 15 , n = 13 C 57 H 107 O 24 , n = 22 C 40 H 63 O 10 , n = 7

532.3490

C 27 H 50 O 9 N, [M + NH 4 ] + C 39 H 74 O 15 N, [M + NH 4 ] + C 57 H 110 O 24 N, [M + NH 4 ] + C 40 H 66 O 10 N, [M + NH 4 ] + C 52 H 90 O 16 , [M + NH 4 ] +

779.4812

779.4787

3.21

796.5080

1175.7157

1175.7147

0.85

1192.7434

IBU PEG diester

703.4432

703.4416

2.27

720.4691

967.5987

967.5989

0.21

C 52 H 87 O 16 , n = 13

984.6274

1363.8367

1363.8348

1.39

C 70 H 123 O 25 , n = 22

 

IBU

sorbitol

371.2068

371.2064

1.08

C

19

H

31

O

7

388.2334

C 19 H 34 O 7 N, [M + NH 4 ] + C 19 H 29 O 6 , [M-H 2 O + H] + C 19 H 27 O 5 , [M-2H 2 O + H] + C 19 H 32 O 6 N, [M + NH 4 ] +

 

353.1962

335.1857

IBU

sorbitan

353.1962

353.1959

0.85

C

19

H

29

O

6

370.2227

 

335.1856

C 19 H 27 O 5 , [M H 2 O + H] +

161.1325

C 12 H 17 , [IBU HCO 2 ] +

Table III. FT-IR Spectral Data for IBU Esters

Impurity

IR

IBU PEG monoester

ν(OH) 3500, ν (CH) 2953, 2867, ν(C = O) 1732, ν(C = C) 1512, 1456, ν (COC) 1201, ν(CO) 1095 ν(OH) 3360, ν (CH) 2951, 2867, ν(C = O) 1702, ν(C = C) 1512, 1462, ν (COC) 1213, ν(CO) 1089, 1024 ν(OH) 3402, ν (CH) 2952, 2918, 2865, ν(C = O) 1723, 1700, ν (C = C) 1512, 1464, ν(COC) 1168, ν (CO) 1064, 1043

IBU

sorbitol

IBU

sorbitan

All frequencies are in per cm.

2,545 binary gradient module, 2,767 sample manager, 515 HPLC pump, MS and 2,487 dual-wavelength absorbance detector; Waters, USA). The ow rate of 20 ml/min was employed throughout the preparation. The injection volume was 900 μl. In order to monitor UV signal from the semi-preparative column, the efuent was splitted in the ratio 1:1,000 into the methanol ow from 515 HPLC pump, which was directed to MS and UV detector. A semi- preparative XBridge Prep C18 OBD column (100 × 19 mm 2 , 5 μm; Waters, USA) was used for preparative purposes. Method IV was used for isolation of IBU PEG monoester:

The fraction collection was tri ggered by setting a minimal inten- sity threshold (MIT = 3,500 μ V for UV detection) of the UV sig- nal at 265 nm. The mobile phase consisted of 0.025% aqueous solution of ammonium hydroxide (solvent A) and acetonitrile (solvent B). The separation was performed using gradient elution based on following program: time/% of solvent B: 0/50, 2.0/50, 4.4/80, 4.6/100, 6.5/100, 7.0/50 with a re-equilibration time of 1.0 min. Method V was used for preparation of IBU sorbitol and IBU sorbitan: The fraction collection was triggered by setting a minimal intensity threshold (MIT = 15,000 μ V for UV detection) of the UV signal at 265 nm. The mobile phase consisted of 0.1% aqueous solution of formic acid (solvent A) and acetonitrile (sol- vent B). The separation was performed using gradient elution based on following program: time/% of solvent B: 0/35, 0.5/35, 7.5/42, 7.7/100, 8.7/100, 8.9/35 with a re-equilibration time of 1.1 min.

NMR instrumentation and methods

Nuclear magnetic resonance (NMR) spectra were obtained using a Bruker Avance 500 (Bruker Biospin, Germany) at 500.13 MHz (1H) and 125.78 MHz (13C) with Prodigy probe in CDCl 3 at 298 K.

Results

Identication of unknown degradation products

Unknown degradation products were detected during the long-term stability testing of the IBU SGCs formulation in climatic chamber at 25° C and 60% relative humidity (Fig. 1). Gradient UHPLC method (Method I) was used showing relative retention time (RRT) 0.78, 0.86, 1.05, 1.521.63 in respect to IBU. The similar impurities pro- le was observed for IBU SGCs developed by Zentiva and obtained from original manufacturer (data not shown). Subsequently, HRAM LC MS measurement was performed in order to investigate chemical structure of the unknown degradants. HPLC peak corresponding to impurity RRT 1.05was identied as ester of IBU and PEG (IBUPEG monoester). PEG is used as a ll of the SGCs and the ratio of IBU with PEG in the formulation is 2:1. The polymeric compound consisted of a varying number of repeating ethoxy units between terminal hydroxyl group and IBU group (Fig. 2A). The isotopic pattern was represented by typical polymeric envelope with distance corresponding to ethylenglycol monomer unit (Δ 44, C 2 H 4 O). Molecular formula was identied as C 2 n + 13 H 4 n + 18 O n + 2 and monoisotopic mass was ranging from 514.3142 (for n = 7) to 1,174.7074 (for n = 22). The range of molecular weight was in agreement with specication of PEG 600, which was used in the formulation. Moreover, degradation product RRT 1.521.63was identied as conjugate of two IBU molecules connected by PEG via ester bonds (IBUPEG diester, Fig. 2B). Molecular formula was deter- mined as C 2 n + 26 H 4 n + 34 O n + 3 and monoisotopic mass was ranging from 702.4343 (for n = 7) to 1,362.8275 (for n = 22). The isotopic pattern was again represented by typical polymeric envelope. Two other degradants arised from reaction of IBU with sorbi- tol and sorbitan, which are comp onents of the shell and serves as plasticizers. Impurity RRT 0.78 was identi ed as IBU sorbitol

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Douš a et al.

monoester (IBUsorbitol, Fig. 2C) with molecular formula C 19 H 30 O 7 and molecular weight 370. The proposed structure for impurity RRT 0.86was IBU sorbitan monoester (IBUsorbitan,

Fig. 2 D) with molecular formula C 19 H 28 O 6 and molecular weight

352.

Synthesis and puri cation of identi ed impurities

Preparation of IBUPEG esters IBUPEG diester was prepared by procedure from Ref. (27). IBU PEG monoester was prepared by modication of procedure ( 27), in which ratio of reactants was changed to the excess of PEG and only one equivalent of dicyclohexylcarbodiimide (DCC) was used (Fig. 3). 1.7 g of IBU (8 mmol), 1.7 g of DCC (8 mmol) and 0.16 g of 4-dimethylaminopyridine (DMAP) were dissolved in 50 ml of di- chloromethane. Solution of 9.6 g of PEG in 50 ml of dichloro- methane was added to the mixture. Mixture was stirred under laboratory temperature for 15 h. Then solid dicyclohexyl urea was ltered off. Clear solution was evaporated to the viscous semicrys- talline matter. The residuum was diluted by 10 ml of acetone and cooled to 18 °C. Obtained solid was ltered off and liquid portion was concentrated under vacuum of 1mbar and 40° C. Remaining 9.5 g was mixture of semisolid IBU, PEG and IBUPEG esters. The product was dissolved in acetonitrile to concentration 50 mg/ml and subsequently puried by semi-preparative HPLC. The collection

parameters of the semi-preparative HPLC method were optimized with respect to high concentration of IBU PEG esters in the solution

of crude sample. The solution of crude sample was injected onto the

semi-preparative column and the fractions of IBUPEG esters were repeatedly collected and combined. The combined fractions were evaporated under vacuum at 40 °C to obtain pure liquid oily stan- dard. Yield was 2.6 g of IBU PEG monoester.

Preparation of IBUsorbitol ester

A 0.91g of D-sorbitol (5 mmol) was dissolved under mild heating in

5 ml of dimethylsulfoxide (DMSO) to form clear colorless solution.

To this warm solution 1.03 g (5 mmol) of IBU and 0.06 g (0.5 mmol)

of DMAP was added. Subsequently, 1.03 g (5 mmol) of DCC was

added. During addition of DCC white precipitate of dicyclohexyl urea started to form. The mixture was stirred for 20 h, and then cooled in liquid nitrogen to solidify it. This solid mixture was placed to lyophili- zator to reduce DMSO amount. After 4 h the content of DMSO was reduced to 3 g. Dichloromethane (15 ml) was added to the mixture, the mixture was cooled down to 5°C and the solid was ltered off. Filtrate was concentrated in vacuum at 50°C to receive 4.13 g colorless oil. This oil was puried using semi-preparative HPLC. Collected frac-

tions containing desired product were evaporated at 40°C under

reduced pressure. Resulting white waxy solid was dried in vacuum of

oil pump. Yield was 0.3 g of IBUsorbitol ester.

Preparation of IBUsorbitan ester

Polysorb 80 (85/70/00) was concentrated in rotary evaporator under reduced pressure at 50 °C to obtain colorless honey, in which 6.6%

of water was found using Karl Fischer titration method. This con-

centrated polysorb was used for synthesis. A 8.94 g of preconcen- trated polysorb 80 was dissolved in 5 ml warm DMSO, and then 4.58 g (28 mmol) carbonyldiimidazole (CDI) was added to the

warm solution in portions. Mixture was foaming due to evolution

of carbon dioxide. To the separately prepared solution of 5.16 g

(25 mmol) IBU in 3 ml DMSO was added 4.05 g (25 mmol) CDI in

(25 mmol) IBU in 3 ml DMSO was added 4.05 g (25 mmol) CDI in Figure

Figure 4. UHPLC chromatogram of a dosage form after stability storage at 25°C and 60% relative humidity for 18 months using Method I (A). Separation of the IBUPEG monoester using Method III (B) and IBUPEG diester using Method II . The percentage of individual oligomeric units is de- picted above each chromatographic peak.

portions. After all CDI had been added, the solution was stirred for 10 min and then slowly added to the solution of polysorb in DMSO. Resulting clear slightly yellow solution was stirred for 60 h. This mixture was puried using semi-preparative HPLC. Collected frac- tions containing desired product were evaporated at 40° C under reduced pressure. Resulting oil was dried in vacuum of oil pump. Yield was 0.4 g of IBUsorbitan ester. Methods of preparation of IBUPEG, IBUsorbitol and IBUsor- bitan esters were not further optimized due to preparation of analyt- ical standards only.

Esterication of Ibuprofen in Soft Gelatin Capsules FormulationsIdentication, Synthesis and Liquid

7

The characterization of reference standards

In order to prepare in-house standards of IBUPEG monoester, IBUsorbitol ester and IBUsorbitan ester, the structures of prepared impurities were conrmed and characterized by NMR, MS and IR analysis (Tables IIII). The assignment of NMR signals was per- formed by means of 1 H, 13 C and 2D (COSY, HSQC and HMBC) spectra. NMR data unambiguously conrmed the proposed struc- tures of IBU esters. According to the intensities of the NMR signals, average number n of PEG units in IBUPEG monoester was 13. Moreover, the elucidation of chemical structure by NMR showed the IBUsorbitol in-house standard is racemic mixture of four isomers, mainly sorbit-1-yl 2-(4-isobutylphenyl)propionate and sorbit-6-yl 2-(4-isobutylphenyl)propionate. Each NMR 1 H and 13 C NMR signal of IBUsorbitan was formed by one major, one medium and 1 2 minor peak due to mixture of four isomers, mainly 1,4-anhydrosorbit-6-yl 2-(4-isobutylphenyl)propionate and 3,6- anhydrosorbit-1-yl 2-(4-isobutylphenyl)propionate. Further stereo- chemical investigation of the formed monoesters was not subject of this study.

Discussion

Identication of unknown degradation products

The routine UPLC method was then optimized to reach separation of IBU PEG oligomers. Previously, the resolution between PEG oli- gomers using LC methods was achieved at elevated temperature using gradient elution ( 30). The routine UHPLC method was

modied by changing the organic solvent (methanol), gradient prole and increasing the temperature to achieve baseline resolution of the individual oligomers. UHPLC methods were used for the chemical characterization of IBUPEG monoesters (Method III) and IBUPEG diesters (Method II). The separation of the corresponding IBUPEG esters is shown in Figure 4, including the evaluation of percentage of individual oligomers in IBUPEG esters by internal normalization. The Method II was successfully applied for determination of related substances in pharmaceutical SGCs formulations.

The characterization of reference standards

The obtained MS data for prepared standards were in agreement with the data measured in IBU SGCs pharmaceutical formulation (Fig. 5 ). UHPLC peak equivalency between synthesized in-house standards and impurities eluting in stability and forced degradation samples was also conrmed (data not shown). Finally, esters were quantitatively characterized for pharmaceuti- cal use and the corresponding in-house reference standards were es- tablished. The potency of the standards was calculated based on the values obtained from determination of impurities (organic, inor- ganic, water and residual solvents) by applying the principle of mass balance. The potency was 97.0% for IBUPEG monoester, 95.3% for IBU sorbitol and 90.9% for IBU sorbitan. The response correc- tion factor was also determined in respect to IBU API for UHPLCUV method at 220 nm, namely 3.7 for IBUPEG monoester, 1.9 for IBUsorbitol and 1.9 for IBU sorbitan.

1.9 for IBU – sorbitol and 1.9 for IBU – sorbitan. Figure 5. Full scan HRAM

Figure 5. Full scan HRAM mass spectra in positive APCI ionization mode of synthesized IBU PEG monoester (A), IBU PEG diester (B, average mass spectrum at RT ranging from 24.0 to 25.9 min), IBUsorbitol (C) and IBU sorbitan (D).

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Douš a et al.

Conclusion

New impurities were identied in IBU SGCs formulation. The analyti- cal standards of impurities were synthesized, puried and fully char- acterized by HRAM LCMS, NMR and IR. All identied impurities arised from drug-excipient interaction. IBUPEG monoester and IBUPEG diester are formed by esterication of IBU with PEG, the ll of the SGC. On the other hand, IBUsorbitol and IBUsorbitan esters are created by reaction of IBU with plasticizer of SGC shell. The results show that despite many SGCs advantages, SGCs for- mulations show also some disadvantages. The stability of an API in a liquid dosage form and compatibility of the excipients are major challenges. Most of the lls and plasticizers which are currently in use contain hydroxyl groups, which might bear a risk for API con- taining carboxylic groups due to possibility of esterication. Finally, fast and efcient UPLCUV method was developed to monitor newly described degradation products and might be helpful for further formulation development of SGCs IBU products.

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