Highly sensitive endotoxin detection using a gold nanoparticle loaded layered molybdenum disulfide-polyacrylic acid nanocomposite.

Endotoxins or lipopolysaccharides (LPS) are pathogens released from the outer membrane of gram-negative bacteria which produce toxic effects on humans. The sensitive and selective detection of LPS is in high demand, especially in the field of medical supplies, therapeutics and in the food industry. Herein we report a new nano-probe based on a gold nanoparticle loaded, water-soluble layered molybdenum disulfide-polyacrylic acid (Au/MoS2-PAA) nanocomposite as a label-free voltammetric aptasensor for ultrasensitive LPS detection. MoS2 nanosheets were obtained through one-step sonication assisted exfoliation of bulk MoS2 with polyacrylic acid (PAA). Au nanoparticles were incorporated into the MoS2-PAA nanocomposite and thiol terminated LPS binding aptamers (LBA) were immobilized on this. The specific binding of LPS with LBA is investigated electrochemically by differential pulse voltammetry. The apparent binding constant (Kb) of LPS with LBA has been calculated to be 1.53 × 102 mL g-1. The aptasensor demonstrated LPS detection down to the ag mL-1 level without incorporating any redox mediator and showed wide linearity from 100 ag mL-1 to 100 pg mL-1 with a low limit of detection of 29 ag mL-1. The sensor showed excellent recovery upon spiking LPS in clinical grade insulin, suggesting that LBA/Au/MoS2-PAA/GCE has promising application for the trace analysis of LPS in the field of pharmaceutical products.

Nondestructive Quantitative Inspection of Drug Products Using Benchtop NMR Relaxometry-the Case of NovoMix® 30.

Batch-level inference-based quality control is the standard practice for drug products. However, rare drug product defects may be missed by batch-level statistical sampling, where a subset of vials in a batch is tested quantitatively but destructively. In 2013, a suspension insulin product, NovoLog® Mix 70/30 was recalled due to a manufacturing error, which resulted in insulin strength deviations up to 50% from the labeled value. This study analyzed currently marketed FlexPen® devices by the water proton transverse relaxation rate using a benchtop nuclear magnetic resonance relaxometer. The water proton transverse relaxation rate was found to be sensitive to detecting concentration changes of the FlexPen® product. These findings support the development of vial-level verification-based quality control for drug products where every vial in a batch is inspected quantitatively but nondestructively.

Different insulin concentrations in resuspended vs. unsuspended NPH insulin: Practical aspects of subcutaneous injection in patients with diabetes.

AIMS: This study measured the insulin concentration (Ins[C]) of NPH insulin in vials and cartridges from different companies after either resuspension (R+) or not (R-; in the clear/cloudy phases of unsuspended NPH). METHODS: Measurements included Ins[C] in NPH(R+) and in the clear/cloudy phases of NPH(R-), and the time needed to resuspend NPH and time for NPH(R+) to separate again into clear/cloudy parts. RESULTS: In vials of NPH(R+) (assumed to be 100%), Ins[C] in the clear phase of NPH(R-) was<1%, but 230±41% and 234±54% in the cloudy phases of Novo Nordisk and Eli Lilly NPH, respectively. Likewise, in pen cartridges, Ins[C] in the clear phase of NPH(R-) was<1%, but 182±33%, 204±22% and 229±62% in the cloudy phases of Novo, Lilly and Sanofi NPH. Time needed to resuspend NPH (spent in tipping) in vials was brief with both Novo (5±1s) and Lilly NPH (6±1s), but longer with all pen cartridges (50±8s, 40±6s and 30±4s from Novo, Lilly and Sanofi, respectively; P=0.022). Time required for 50% separation into cloudy and clear parts of NPH was longer with Novo (60±7min) vs. Lilly (18±3min) in vials (P=0.021), and affected by temperature, but not by the different diameter sizes of the vials. With pen cartridges, separation into clear and cloudy parts was significantly faster than in vials (P<0.01). CONCLUSION: Ins[C] in NPH preparations varies depending on their resuspension or not. Thus, subcutaneous injection of the same number of units of NPH in patients with diabetes may deliver different amounts of insulin depending on its prior NPH resuspension.

Is insulin diluted when stored in water?

BACKGROUND: Insulin storage is a challenge in resource-poor countries. In Uganda, patients were noted to store insulin vials by submerging them in water. OBJECTIVE: To examine whether withdrawing insulin from a vial without adding air back causes a vacuum which allows water to enter the vial, resulting in insulin dilution. METHODS: Seven hundred units of insulin were withdrawn from forty 10 mL vials of 100 units/mL insulin [20 neutral protamine hagedorn (NPH), 20 regular]. In half, air was added back. The vials were weighed (baseline). Half of the vials (10 with added air, 10 without) were submerged in water for 24 h and then air-dried for 24 h. Vials that were not submerged sat at room temperature for 48 h. All vials were weighed 48 h from baseline. RESULTS: Addition of air did not impact the change in weight after submersion (air added: -0.002 ± 0.001 g or -0.2 ± 0.1 unit; no air added: -0.003 ± 0.000 g or -0.3 ± 0 unit, p = 0.57). In a subset of vials in which an additional 240 units were withdrawn before submersion for another 24 h, there was still no difference in weight change in those vials with air added (p = 0.2). CONCLUSION: Withdrawing insulin from a vial without adding air did not result in uptake of water or dilution of insulin in the submerged vial, although it made drawing up the insulin easier. This study did not address the larger concern of bacterial contamination of the rubber stopper during water storage.

Characteristics of commercially manufactured and compounded protamine zinc insulin.

OBJECTIVE: To evaluate and compare characteristics of a commercially manufactured protamine zinc insulin (PZI) product and PZI products obtained from various compounding pharmacies. DESIGN: Evaluation study. SAMPLE: 112 vials of PZI (16 vials of the commercially manufactured product and 8 vials from each of 12 compounding pharmacies) purchased over an 8-month period. PROCEDURES: Validated methods were used to analyze 2 vials of each product at 4 time points. Appearance, endotoxin concentration, crystal size, insulin concentration in the supernatant, pH, total insulin and zinc concentrations, and species of insulin origin were evaluated. RESULTS: All 16 vials of commercially manufactured PZI met United States Pharmacopeia (USP) specifications. Of 96 vials of compounded PZI, 1 (1 %) contained a concentration of endotoxin > 32 endotoxin U/mL, 23 (24%) had concentrations of insulin in the supernatant > 1.0 U/mL, and 45 (47%) had pH values < 7.1 or > 7.4; all of these values were outside of specifications. Several vials of compounded PZI (52/96 [54%]) did not meet specifications for zinc concentration (0.06 to 0.1 mg/mL for 40 U of insulin/mL, 0.075 to 0.12 mg/mL for 50 U of insulin/mL, and 0.15 to 0.25 mg/mL for 100 U of insulin/mL), and total insulin concentration in 36 [38%] vials was < 90% of the labeled concentration. CONCLUSIONS AND CLINICAL RELEVANCE: Only 1 of 12 compounded PZI products met all USP specifications in all vials tested. Use of compounded PZI insulin products could potentially lead to serious problems with glycemic control in veterinary patients.

Assessment of the mixing efficiency of neutral protamine Hagedorn cartridges.

Reliable application of neutral protamine Hagedorn (NPH) insulin requires previous resuspension of the suspension by tipping over the cartridge 20 times. This procedure is considered annoying by patients. The goal of this investigation was to assess the efficiency of the mixing procedure when performed less frequently than recommended. Neutral protamine Hagedorn insulin cartridges from five different manufacturers (sanofi-aventis, Lilly, Berlin-Chemie, B. Braun, and Novo Nordisk) were emptied with doses of 28 IU in the morning and the evening over 5 days. While the first dose was obtained after a regular resuspension procedure (20x tipping over), the consecutive doses were obtained after 3, 6, 10, or 20 mixing procedures (12 cartridges per experimental series, two doses/day). Insulin concentrations of doses 1, 2, 6, and 10 were determined by high-pressure liquid chromatography. Between dosing, cartridges were stored at room temperature in a horizontal position. Comparable insulin concentrations were seen in the first correctly prepared doses. Pronounced and substantial deviations from the selected dose were observed with most of the cartridges, in particular when resuspending only 3 and 6 times. Mean absolute percentage deviations when tipping 3 times and maximally observed overdoses were: Insuman basal: 1.1 +/- 1.0%/4 IU, Humulin N: 2.6 +/- 3.4%/19 IU, Berlinsulin H basal: 4.4 +/- 6.0%/26 IU, Insulin B. Braun basal: 10.4 +/- 8.9%/38 IU, and Protaphane: 4.7 +/- 4.1%/19 IU (all p < 0.05 vs Insuman basal). Only one cartridge with three metal mixing bullets (sanofi-aventis) was resuspended efficiently with only a few mixing procedures. All other cartridges with fewer bullets were shown to deliver potentially harmful doses if used for treatment when the mixing procedure was less frequent than demanded in the instructions for use.

An analysis of the mixing efficiency of neutral protamine Hagedorn cartridges.

In this issue of Journal of Diabetes Science and Technology, Kaiser and colleagues conducted an investigation to identify variations in the delivered dose of several different isophane insulin (neutral protamine Hagedorn, NPH) brands that use glass and metal bodies ("bullets") to facilitate mixing. Using a strategy where multiple pens from each of five different NPH insulin products (Insuman Basal, sanofi-aventis, three metal bullets; Humulin N, Lilly, one glass bullet; Berlinsulin H Basal, Berlin-Chemie, one glass bullet; Insulin B. Braun Basal, two glass bullets; and Protaphane Penfill, NovoNordisk, one glass bullet) were compared at multiple sampling points and over a range of mixing procedures (3, 6, 10, and 20 times), the authors identified deviations in the delivered dose of insulin at initial use and with repeated dosing. At the initial dose, adhering with manufacturer recommendations to conduct the mixing procedure 10-20 times was found to demonstrate minimal deviation and there was no pronounced difference among the products. Decreasing the number of mixing procedures from 10-20 to 3-6 times, a more profound deviation was noted, with the Insuman Basal product demonstrating less variability in comparison to all other products evaluated. A repeated dose study (1, 2, 6, and 10) with only six mixing procedures revealed that the insulin concentration of each dose increased for all products except Insuman Basal. Clinically, numerous factors may contribute to variability observed with subcutaneous administration of isophane insulin. While data presented by Kaiser and colleagues demonstrated that the issue of proper mixing is not trivial, the modest differences observed between and within products both at the initial dose and with repeated dosing may indicate that the clinical relevance of these findings is most applicable to those requiring large doses or, alternatively, those who have otherwise unexplained hypoglycemic episodes.

Beyond the era of NPH insulin--long-acting insulin analogs: chemistry, comparative pharmacology, and clinical application.

The new rDNA and DNA-derived "basal" insulin analogs, glargine and detemir, represent significant advancement in the treatment of diabetes compared with conventional NPH insulin. This review describes blood glucose homeostasis by insulin in people without diabetes and outlines the physiological application of exogenous insulin in patients with type 1 and type 2 diabetes. The requirements for optimal basal insulin treatment are discussed and the methods used in the evaluation of basal insulins are presented. An essential criterion in the development of an "ideal" basal insulin preparation is that the molecular modifications made to the human insulin molecule do not compromise safety. It is also necessary to obtain a clear understanding of the pharmacokinetic and pharmacodynamic characteristics of the two currently available basal insulin analogs. When comparing glargine and detemir, the different molar concentration ratios of the two insulin formulations should be considered along with the nonspecificity of assay systems used to determine insulin concentrations. However, euglycemic clamp studies in crossover study design provide a good basis for comparing the pharmacodynamic responses. When the latter is analyzed by results of intervention clinical trials, it is concluded that both glargine and detemir are superior to NPH in type 1 and type 2 diabetes. However, there is sufficient evidence to demonstrate that these two long-acting insulin analogs are different in both their pharmacokinetic and pharmacodynamic profiles. These differences should be taken into consideration when the individual analogs are introduced to provide basal insulin supplementation to optimize blood glucose control in patients with type 1 and type 2 diabetes as well. PubMed-Medline was searched for articles relating to pharmacokinetics and pharmacodynamics of glargine and detemir. Articles retrieved were reviewed and selected for inclusion if (1) the euglycemic clamp method was used with a duration >or=24 h, (2) a single subcutaneous dose of glargine/detemir was used, and (3) area under the curve for insulin concentrations or glucose infusion rates were calculated.