APPENDIX 1 - Lippincott Williams & Wilkins



APPENDIX 1

A benzodiazepine assay was developed to quantify the plasma concentrations of lorazepam and midazolam from patient plasma in order to compare the pharmacokinetics of the two drugs under conditions of constant infusion. New extraction and HPLC methods were developed by adapting techniques from previous studies1-3 and were validated according to suggested guidelines.4

The drugs were extracted from plasma using 1000 ng(mL-1 diazepam in 0.1 mM HCl as the internal standard. One mL of the internal standard solution and 1.0 mL patient plasma were placed in a 15 mL screw cap Pyrex test tube and 1.0 mL of a saturated sodium borate solution, pH 7.0 was added to it. Extraction was performed twice with 5 mL of butylchloride (1-chlorobutane), after which the butylchloride was evaporated. The residue was then reconstituted in 100 (L of the HPLC running Buffer A. The running buffers were made with sodium acetate (10 mM, pH 4.6): methanol: acetonitrile in a ratio of 375:300:325 (Buffer A) and 0:500:500 (Buffer B). The samples were eluted at a 1.0 mL(min-1 flow rate through a 3.9 x 150 mm Waters SYMMETRY C18 (Millipore Corp., Milford, MA), 5 (m reverse-phase column at 30(C. The assay was performed on a HP 1050 HPLC (Hewlett Packard, Palo Alto, CA, USA) using a Diode Array Detector with HP 3D ChemStation software. Isocratic elution of the drugs of interest was performed with Buffer A for 6 min. A linear gradient to 100% Buffer B occurred over 2 min; 100 % Buffer B was maintained for 10 min to wash the column. And then a linear gradient to 100% Buffer A occurred over 3 min. Buffer A was maintained at 100 % for 11 min to prepare the column for the next injection. Detection was made at 230 ( 5 nm wavelength for lorazepam, midazolam, and diazepam with a reference wavelength at 550 ( 100 nm.

With each set of calibration curve samples, a sample of blank plasma was extracted and run to observe background interference. This blank spectrum was subtracted from the spectrum of the spiked samples. The calculations for drug concentration are based on a linear standard curve in the 1000 to 16 ng(mL-1 plasma range (1000, 500, 250, 125, 62, 31, 16). The correlation coefficients for the curves were 0.9954 and 0.9959, respectively. Duplicate samples of a quality control (QC) spiked serum at a low, medium, and high concentration (31, 125, 500 ng(ml-1 of lorazepam and midazolam) were run at the beginning of each set of samples. Plasma was then extracted and assayed to determine % recovery: lorazepam 71.2%, midazolam 102.8%, and diazepam 104.1 %. Absolute recovery was determined over the entire validated concentration range: lorazepam 71.4 %, midazolam 94.1 %, and diazepam 96.2 %. It was anticipated that fentanyl and intralipid would be co-administered during clinical trials. It was established that fentanyl elutes well before the analytes, and that blood was obtained from two patients receiving intralipid did not show interfering peaks. Equipment (needles, catheters, pipettes, collection jars, etc.) which came into contact with the physiological fluid during collection or storage was shown not to interfere with, or absorb, the analytes of interest.

In these experiments, duplicate pairs of assays agreed within: blank, 6%; lorazepam, 3%; and midazolam, 5%. The limit of quantitation where the relative standard deviation (RSD) is SS (2-6); C50,ss = plasma benzodiazepine concentration where [pic] = 50%; ( = slope of probability curve; CB = plasma benzodiazepine concentration; CF = plasma fentanyl concentration; FA = additive fentanyl effect; FS = synergistic fentanyl–benzodiazepine effect; VD = virtual drug effect parameter; K = VD elimination rate constant, t = time since end of anesthesia, (1 = C50,ss of midazolam (SS = 2-6); (2 = lorazepam potency parameter; (3 = fentanyl effect parameter; (4 = age effect parameter; and Lorazepam flag and Fentanyl flag = 0 or 1 depending on whether or not the drug is present. In model H only, the parameter ( = [(lorazepam ( Lorazepam flag + (midazolam ( (1-Lorazepam flag)]. Got it?

Appendix Table 2. Typical values for the original and revised estimated pharmacokinetic parameters for lorazepam and midazolam.

| |Lorazepam |Midazolam |

|Model Parameters |Greenblatt et al11 |Present study |Zomorodi et al18 |Present study |

| | |(n = 14) | |(n = 9) |

|Volumes: (L) | | | | |

|Central (V1) |32.2 * |40.8 |33 |28.2 * |

|Peripheral (V2) |41.3 * |102 |64.3 * |131 |

|Slow peripheral (V3) |--- |--- |365 |--- |

|Vss |73.5 |142.8 |462.3 |159.2 |

| | | | | |

|Clearances: (L(min-1) | | | | |

|Metabolic (CL1) |0.07 * |0.107 |0.38 * |0.25 * |

|Peripheral (CL2) |3.01 * |1.86 |0.622 |0.599 |

|Slow peripheral(CL3) |--- |--- |0.264 |--- |

| | | | | |

|Fractional coefficients: | | | | |

|A |0.57 |0.731 |0.86 |0.896 |

|B |0.43 |0.269 |0.12 |0.104 |

|C |---- |---- |0.016 |---- |

|Exponents: (min-1) | | | | |

|( |0.166 |0.0657 |0.044 |0.033 |

|( |0.0009 |0.00073 |0.0045 |0.0012 |

|( |---- |---- |0.0004 |---- |

|Half-lives: (min) | | | | |

|( |4.14 |10.55 |15.81 |20.71 |

|( |736 |952.52 |153.4 |572.3 |

|( |---- |---- |1704 |---- |

|Rate constants: (min-1) | | | | |

|k10 |0.002 |0.00262 |0.012 |0.009 |

|k12 |0.093 |0.04559 |0.0188 |0.021 |

|k13 |---- |---- |0.008 |---- |

|k21 |0.072 |0.01824 |0.0097 |0.005 |

|k31 |---- |---- |0.0007 |---- |

| | | | | |

|Performance measures: | | | | |

|MDWR (%) |-24.38 |-2.6 |23.56 |5.39 |

|MDAWR (%) |28.92 |17.27 |43.25 |22.01 |

*Pharmacokinetic parameters for a 65 yr old male, wt. = 70 kg, ht. = 180 cm, BSA = 1.9 m2

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