Clinical Communications Penicillin stability in prefilled syringes for the purpose of skin testing for drug allergy Alka Garg, PhD a , Damien Chan, MBBS, DCH, FRACP b , Fotios Ambados, BPharm c , Ei Lwin, BPharm d , Yunmei Song, PhD e , and Sanjay Garg, PhD f Clinical Implications  Dilutions of penicillin class drugs in syringes, as required for skin testing for drug allergy, are stable for 2 to 7 days. This study supports a change in clinical practice where reagents for penicillin skin prick tests and intradermal tests can be prepared in advance by pharmacy, improving safety and workflow. TO THE EDITOR: Up to 10% of the patients attending hospitals report an allergy to penicillin, though only about 6% to 10% of these have a true IgE-mediated allergy, proven by cutaneous tests and drug prov- ocation. 1,2 A label of penicillin allergy when a true allergy is not present often leads to lifelong avoidance of penicillin and related drugs. 3 This leads to the use of less specific, more expensive antibiotics, contributing to increased adverse effects and antibi- otic resistance. The negative predictive value for penicillin skin testing for immediate reaction is close to 100%, whereas the positive pre- dictive value is between 40% and 100%. 4 Diagnosis is currently confirmed using cutaneous tests (skin prick testing [SPT] and intradermal testing [IDT]) with penicilloyl poly-L-lysine (PPL) as the major determinant of benzylpenicillin and minor determi- nant mixture (MDM) containing benzylpenicillin, benzylpeni- cilloate, and benzylpenilloate. 5,6 SPT and IDT, using penicillin dilutions and PPL and MDM, are widely recommended for testing immediate hypersensitivity to penicillin class antibiotics. 7 In Europe, both PPL and MDM are available with Diater DAP (Madrid, Spain). In the United States, only PPL is available as PRE-PEN (AllerQuest LLC, Plainville, Conn). Traditionally, test reagents, including drug dilutions and allergic determinants, are made at the bedside by the allergist because there is no evidence of their stability in the syringes these are prepared in. This is a time-consuming, labor-intensive practice and limits the number of patients who can be tested at one time. Also, with multiple antibiotics, at multiple dilutions, errors can be made. If these dilutions could be prepared in advance in pharmacy under aseptic conditions and labeled appropriately, it would not only improve safety of testing but also contribute significantly to improving the workflow of allergy testing clinics and facilitate more patients being tested. There would be financial gains in terms of less drug wastage and time saved in preparing syringes for multiple patients together rather than one at a time. Table I 8 details the reagents used for SPT for penicillin class antibiotics. Other drugs can be included on the basis of individual patient requirements. Pharmacy at Women’s and Children’s Hospital in Adelaide makes these dilutions within 24 hours of testing, so that patients do not have to wait while these dilutions are being made. However, there is no literature regarding the stability of these dilutions in the syringes in which they are prepared in advance. Recent information from Diater (available in Australia) confirms the stability of PPL and MDM after reconstitution to be 15 days in refrigerated conditions (Sanchez J, 2013; unpublished data). The aim of this study was to carry out validated stability testing of penicillin class drugs commonly used for SPT and IDT in syringes to enable their preparation before the tests. HPLC analytical methods were developed for benzylpenicillin sodium (Penicillin G sodium salt, Sigma, Sydney, Australia), amoxicillin sodium (Amoxil, GlaxoSmithKline, Uxbridge, UK), and flucloxacillin sodium (DBL Flucloxacillin sodium, Hospira Australia, Melbourne, Australia) to ensure that they were adequate and repeatable. The assay methods were validated for linearity, specificity, precision, and stability in the analytical solution. Analysis was carried out using HPLC (Shimadzu, Kyoto, Japan) and Phenomenex Kromasil C-18 column. The mobile phases included acetonitrile, potassium dihydrogen phosphate, and sodium hydroxide for amoxicillin HPLC analysis; methanol, potassium dihydrogen phosphate, and orthophosphoric acid for benzypenicillin HPLC analysis; and acetonitrile, potassium dihydrogen phosphate, and sodium hydroxide for flucloxacillin HPLC analysis. Dilutions of benzylpenicillin sodium (0.6 mg/mL, pH 7.06, and 6 mg/mL, pH 6.98), amoxicillin sodium (2 mg/mL, pH 8.6, and 20 mg/mL, pH 8.66), and flucloxacillin sodium (0.5 mg/mL, pH 6.05, and 5 mg/mL, pH 5.24) were aseptically prepared in sterile water for injection and packed in 1-mL Leur- lock polycarbonate syringes with “Combi” syringe caps. This was done in a sterile pharmacy manufacturing facility by aseptically validated pharmacists. Water for injection was used to further dilute to the required concentrations. Three batches for each dilution were prepared, and 2 sample replicates were drawn from each batch for each sampling point and each temperature. The syringes were transferred to the analytical testing labo- ratory immediately postproduction, maintaining the cold chain, and placed in stability chambers (Newtronic, Mumbai, India) at 25  C, 4  C, and 20  C (Refrigerator/freezer; Samsung, Seoul, South Korea). Samples were withdrawn at different time points ranging from 0 to 7 days 9 and assessed for physical (color change, precipitation) and chemical (drug recovery 90% by HPLC 10 ) stability. No color change or precipitation was observed for the 3 penicillins tested at all temperatures throughout the stability study. All drug dilutions were thereby deemed physically stable at all temperatures tested. Figure 1, A to F, depicts the chemical stability of the tested penicillins (percent drug recovery with respect to time). Based on these results, new shelf life was determined for the dilutions in syringes of the penicillin class antibiotics tested, as detailed in Table II. 1