Jingxian Qi,Qingfen Liu*,Huizhou Liu*

CAS Key Laboratory of Green Process and Engineering,Institute of Process Engineering,Chinese Academy of Sciences,Beijing 100190,China

Keywords:Penicillin G Ionic liquids Stability Rearrangement Kinetics

A B S T R A C T The extraction of penicillin G by ionic liquid[Bmim]PF6 has exhibited promising prospect.The stability of penicillin G is crucial for developing a green ionic liquid-based extraction technology.In this work,the stability of penicillin G in[Bmim]PF6 was systematically investigated.The results showed the stability of penicillin G was significantly influenced by pH and temperature.It tended to be more stable when pH value increased from 1.5 to 4.0 and the temperature gradually decreased.The half-life(t1/2)of penicillin G in[Bmim]PF6 was 17.7 h in the optimal technological condition(p H 2.0 and 10°C),which is enough for the requirement of extraction technology.The reaction of penicillin Gin[Bmim]PF6 followed the first order kinetics in the pH range 2.0–4.0.Three isomers of penicillin G were found through rearrangement at pH 2.0,and their structures were not affected by temperature.

1.Introduction

Penicillin G is the largest production of bulk pharmaceutical chemicals.Its industrial separation has been implemented by extraction with butyl acetate at pH 1.8–2.2[1,2].How ever,this process faces several problems,such as explosion risk,VOC pollution,and severe emulsification[3,4].Therefore,novel environment-friendly extraction processes are highly demanded.

Ionic liquids(IL),as green alternative solvents[5,6],arose increasing attention due to their excellent performance[7,8].Several ILs have been adopted in penicillin extraction[9–12].According to our previous work,when the hydrophobic ionic liquid[Bmim]PF6was used as extraction solvent,the one-stage extraction efficiency reached 91%at the optimal condition of pH 1.5–2.5,T=10 °C,and penicillin G concentration 3.00–5.00 × 104U·ml?1.The emulsification problem mentioned before can be overcome[4].The extraction of penicillin G by[Bmim]PF6has shown promising prospect for industrial application.

The stability of penicillin G is crucial for developing a green ionic liquid-based extraction technology.Penicillin G contains a labile βlactam ring which is easily opened in acidic and alkaline conditions,resulting in impurities generated.Moreover,the stability of penicillin G varied in different solvents.It has been reported that penicillin G is unstable in acidic aqueous solutions[13,14],the half time of penicillin G in water is 1.3 h at pH 2.0 and 10°C.After extraction,penicillin G stayed into butyl acetate and its stability was improved.The reaction of penicillin G was hardly observed in butyl acetate after 24 h at 10°C[15].

Until now,the stability study of penicillin G in[Bmim]PF6has not been reported in the literature.The stability of penicillin G in[Bmim]PF6must satisfy the extraction duration.Furthermore,the reaction product of penicillin G could affect the separation and severely impact environment[16],e.g.,its antibacterial activity may induce drug-resistant bacteria and super-bacteria[17,18].

In this work,the stability of penicillin G in[Bmim]PF6and the key influencing factors were explored.The reaction of penicillin G in[Bmim]PF6and its kinetics were investigated,too.Moreover,the reaction products were proposed according to analysis results.The results of this work provide the fundamental information for penicillin G green extraction and will help for environmental impact assessments.

2.Materials and Methods

2.1.Materials

Penicillin G potassium(99.5%)was kindly provided by North China Pharmaceutical Group,Shijiazhuang,China.[Bmim]PF6was purchased from Shanghai Chemical Reagents Co.Ltd.with mass fraction purity＞99%.H2SO4(analytical grade)was used in experiment.

2.2.Extraction of penicillin G

Penicillin G solution(3.00 × 104U·ml?1)was prepared by dissolving a certain amount of potassium salt of penicillin G in deionized water.Penicillin G solution and[Bmim]PF6were added into a beaker at avolumeratio of 2:1,then magnetically stirred at the desired temperature for 5 min to reach extraction equilibrium.10%H2SO4(w/v)was used for pH adjustment.Two phases were separated under centrifugation(3000 r·min?1,2 min).Volumes of both phases were measured with measuring cylinders and pH value was recorded.

2.3.Analysis

HPLC(Agilent Technologies 1260 Infinity)was used to analyze penicillin G and reaction products in[Bmim]PF6under 254 nm using a Zorbax SB-C 18 column(5 μm,4.6 mm × 150 mm)at 25 °C.The mobile phase consisted of a methanol stream and an aqueous solution of 10 mmol·L?1ammonium acetate with the gradient going from 30%methanol to 95%methanol in 25 min.The total flow rate was 1 ml·min?1.The injection volume was 10 μl for each sample.

Measurements of molecular weights and structures were made on HPLC(UltiMate 3000)coupled to a mass spectrometer(MS)(Thermo Scientific Q-Exactive).The mobile phases used for this HPLC were methanol and 10 mmol·L?1ammonium acetate.Chromatographic separation was achieved at a flow rate of 0.1 ml·min?1using a gradient as follows:during the initial 2 min the ratio was 30:70(Vmethanol:Vammoniumacetate),went up to 95:5 from 2 to 50 min,then declined to the initial ratio of 30:70 from 50 to 60 min,and this ratio was maintained for additional 10 min.A Thermo Hypersil GOLD C18 column(3 μm,2.1 × 100 mm)of temperature 25 °C was adopted.The injection volume was 5 μl for a sample.

3.Results and Discussion

3.1.Changes of penicillin G in[Bmim]PF6

Samples were taken from[Bmim]PF6with penicillin G dissolved at intervals and analyzed by HPLC.According to Fig.1,penicillin Gchanged in[Bmim]PF6at 20°C and three unknown peaks appeared in HPLC curves.As Fig.1a show s,the peaks at retention time(Rt)of 6.790 min,2.114 min,3.009 min and 11.445 min represented penicillin G and three unknown products—A,B,and C,respectively.

3.2.Effects of pH and temperature on stability

The remaining concentration of penicillin G in[Bmim]PF6versus time was plotted in Fig.2.Penicillin G was much more stable at pH 4.0,at which the concentration of penicillin G slightly decreased(Fig.2b).Compared with pH 4.0,penicillin G decreased faster at pH 2.0,especially at 24°C(Fig.2a).High pH value and low temperature can decelerate the decomposition of penicillin G.However,high pH was not of benefit to make the extraction process of excellent performance.Therefore,low temperature was recommended as a strategy to improve penicillin Gstability.The results showed that p Hand temperature could greatly affect the stability of penicillin G.

Samples of penicillin G in[Bmim]PF6were analyzed by HPLC after keeping at different temperatures for 8 h(Fig.3).The retention time of each peak was almost the same.The results demonstrated that the change of temperature from 10 °C to 30 °C influenced the reaction rate of penicillin Gin[Bmim]PF6but did not generated any new product.

3.3.Reaction kinetics

The relationship of logarithm of concentration and reaction time is usually used to express reaction kinetics.The logarithm of penicillin G concentration versus reaction time was showed in Fig.4.A linear relation was achieved no matter under pH 2.0 or pH 4.0,which follows the first-order kinetic equations:

Fig.1.Changes of penicillin G in[Bmim]PF6.

Fig.2.Effects of pH and temperature on the stability of penicillin G in[Bmim]PF6.

Fig.3.HPLC analysis of penicillin G in[Bmim]PF6 after 8 h at different temperatures.

where C0is the initial concentration of penicillin G and C is the instantaneous concentration at time t(min);k is the rate constant(min?1)which can be calculated from

where m is the straight slope.Similar kinetic pattern of penicillin reaction in acidic media was reported by Blaha et al.[19].

Fig.4.Reaction kinetics of penicillin G in[Bmim]PF6 at 10°C.

3.4.Half-time of penicillin G

The relationship between half-time(t1/2)and k is

Fig.5.Effects of pH and temperature on half life time of penicillin G.

The t1/2values under various pH were calculated using the equations above and were plotted in Fig.5.When pH was in the range of 1.5–4.0,t1/2kept growing with the increase of pH value and with the decrease of temperature,indicating the increase of stability of penicillin G.When pH was in the range of 1.5–2.5,t1/2increased faster at 10 °C than at 24°C.Therefore,reducing temperature was an effective strategy to improve penicillin G stability.

Table 1 lists t1/2values under different solvents.Penicillin G reacts rapidly at pH 2.0 and 24 °C in aqueous solution,and even at 10 °C its half time is just 1.3 h[13],which suggests penicillin G should be quickly transferred out from water.Compared to butyl acetate,t1/2of penicillin G in[Bmim]PF6is shorter,17.7 h in pH 2.0 at 10°C.How ever,this value already satisfies the operation demand in the extraction technology.

Table 1Half life time of penicillin G in different solvents(p H 2.0)

3.5.Reaction product structure

The molecular weights of the three reaction products were measured by HPLC–MS.According to the analysis results,the molecular weights of these products were found to have the same value—334,and they shared the same molecular formula—C16H18N2O4S.They were all isomers of penicillin G.HPLC–MS/MS was applied for structure analysis.The common isomers of penicillin G are iso-penillic acid,penillic acid,penillonic acid,and penicillenic acid.Software Mass Frontier 7.0 was adopted to calculate probable fragment ion peaks generated from the compounds mentioned above.Then the results from calculations were compared with HPLC–MS/MS results to identify the products—A,B and C.All the top seven peaks of product A from HPLC–MS/MS results(at m/z 335.11,301.12,289.10,203.08,176.07,159.09,and 91.05)matched the analysis of iso-penillic acid by Mass Frontier 7.0.How ever,penillic acid,penillonic acid and penicillenic acid just generated six peaks among the seven.Penillic acid and penillonic acid did not generate the peak at m/z 159.09,and penicillenic acid did not generate the peak at m/z176.07.Therefore,product Aisisopenillic acid.Both iso-penillic acid and penillic acid generated all top six peaks of product B(at m/z 335.11,289.10,176.07,160.04,100.04 and 91.05),but penillonic acid did not generate the peak at m/z 100.04 and penicillenic acid did not generate the peak at m/z176.07.Moreover,iso-penillic acid should have characteristic peaks at m/z 301.12 and 159.09.Thus product B is penillic acid.Product C was proposed to be penillonic acid,because the top 12 peaks from HPLC–MS/MS results(at m/z 335.11,317.1,220.04,217.06,203.08,189.07,176.07,160.04,116.07,98.06,91.05 and 70.07)matched its analysis from Mass Frontier 7.0.Iso-penillic acid did not generate peaks at m/z 220.04,189.07,116.07,98.06 and 70.07;penillic acid did not generate peaks at m/z 217.06 and 189.07;and penicillenic acid did not generate peaks at m/z 220.04,176.07 and 116.07.These three rearrangement products are illustrated in Fig.6.

3.6.Possible rearrangement process

Three new peaks emerging at 2.114 min,3.009 min and 11.445 min were observed(Fig.1).They represented three unknown products—A,B,and C.At the beginning of detection,products A,B and C had already existed.As detection time extended,the amount of penicillin G continuously decreased and contents of A and B gradually increased during 48 h of standing at 20°C.What was interesting,the amount of product C increased at first,but kept declining after 24 h.After 48 h,the peak indicating penicillin G nearly disappeared and the product C existed in a small amount.

Fig.6.Mass spectra analysis of rearrangement products.A:iso-penillic acid;B:penillic acid;C:penillonic acid.

A scheme of penicillin G rearrangement in[Bmim]PF6was proposed in Fig.7.It is well-known that the β-lactam of penicillin G is unstable and can be easily opened through acid-catalyzed hydrolysis at pH 2.0.Moreover,the hydrolysis is incomplete,resulting in penicillin G rearrangement to form penillic acid.And penillic acid can be further converted into iso-penillic acid[20,21].Penillonic acid was reported as a possible reaction product in literature,and its rearrangement is also a known rearrangement of penicillin G[22,23].As mentioned in previous paragraphs that,the content of product C—penillonic acid rose and then declined,it can be possibly explained that penillonic acid accumulated at first and was converted into penillic acid.This process was partially different from what happened in acidic aqueous solution,in which penicillenic acid,penillic acid,penamaldic acid,penilloic acid and penicillamine were successively detected.Penilloic acid and penicillamine were proposed as the final products[19].

Fig.7.A proposed rearrangement scheme of penicillin G in[Bmim]PF6.

4.Conclusions

It was found that the stability of penicillin Gin[Bmim]PF6was significantly influenced by p Hand temperature,and it was improved with the increase of pH from 1.5 to 4.0 and with the decrease of temperature.The half life time of penicillin G was 17.7 h at pH 2.0 and 10°C,which met the technological demand of extraction.The reaction of penicillin G in[Bmim]PF6followed the first order equation no matter the extraction was conducted at pH 2.0 or 4.0.Three rearrangement products of penicillin G in[Bmim]PF6at pH 2.0 were identified according to the comparison of results between HPLC–MS and Software Mass Frontier 7.0.These products were found to be isomers of penicillin G:iso-penillic acid,penillic acid and penillonic acid.