THE EFFECTS OF HEME FROM HEMOLYZED MATRIX ON THE STABILITY OF OCTREOTIDE IN K2 EDTA PLASMA. William Arthur Altizer

Transcription

1 THE EFFECTS OF HEME FROM HEMOLYZED MATRIX ON THE STABILITY OF OCTREOTIDE IN K2 EDTA PLASMA William Arthur Altizer A Thesis Submitted to the University of North Carolina Wilmington in Partial Fulfillment of the Requirements for a the degree of Master of Science Department of Chemistry and Biochemistry University of North Carolina Wilmington 2012 Approved by Advisory Committee John Tyrell Scott Wright James Reeves Chair Accepted by Dean, Graduate School

2 TABLE OF CONTENTS ABSTRACT... iii ACKNOWLEDGEMENTS... iv LIST OF TABLES...v LIST OF FIGURES... vi INTRODUCTION...1 METHODS...6 RESULTS AND DISCUSSION...30 CONCLUSIONS...60 LITERATURE CITED...62 ii

3 ABSTRACT An issue during blood collection for drug analysis is that the membranes of the red blood cells can be damaged and leak their contents into the collected specimen. Hemoglobin, one of the components of red blood cells, is comprised of four globular protein subunits and inside each subunit is a protein chain tightly associated with a non-protein heme group. The free heme compounds or free iron Fe (II) from the ruptured red blood cells could interact with other components found in the collected specimen. This research was done to see if the drug octreotide would be affected by the presence of hemolyzed matrix and in particular heme. The results of this research showed that in the plasma treated with the anticoagulant dipotassium ethylenediaminetetraacetate (K2 EDTA) octreotide was stable, for up to three hours, when hemolyzed matrix or heme was added to a prepared sample. The research also looked at the stability of octreotide in solutions containing certain metals. The metals aluminum, iron, and zinc reduced the amount of recovered octreotide. iii

4 ACKNOWLEDGEMENTS I want to extend my thanks to my wife Tamara who has helped to keep me going during the process of working towards my Masters Degree. To my parents and my son, Liam, thank you for being so supportive during these past few years. To Pharmaceutical Product Development, LLC (PPD) I want to say that I appreciate the opportunity that they have opened up for their employees to give them the chance to further their education. Also to Dr. Scott Wright who has been my mentor for the program at PPD I want to extend my thanks for the encouragement and guidance. To Dr. John Tyrell thank you for your support and encouragement during my time in the program. I really appreciate your dedication to seeing that the students in the program are successful in not only completing the program, but asking feedback so that that the program can better serve future students. I also want to thank the University of North Carolina Wilmington for putting forth a distance education program for Master of Science degree in chemistry. Without this wonderful opportunity I may never have found the time to continue my education. iv

5 LIST OF TABLES Table Page 1. The high pressure liquid chromatography gradient program used for the analysis of octreotide samples The high pressure liquid chromatography gradient program used for the second octreotide sample analysis method The standard electrode potentials of aluminum, copper, iron, and zinc A fourfold dilution of an octreotide sample in four different matrix combinations A serial dilution of samples with octreotide and octreotide internal standard to eliminate hemolyzed whole blood matrix effects on solid phase extraction Incubation of octreotide in matrix for 0 and 3 hours at room temperature and 37⁰C Incubation of octreotide IS in matrix for 0 and 3 hours at room temperature and 37⁰C Octreotide and octreotide internal standard samples were treated with hemin, heme, sodium dithionite, Fe II or Fe III to show that octreotide and its internal standard are stable during a 1 hour room temperature incubation with these compounds ng/ml octreotide in 20:80 acetonitrile/water with 0.1% formic acid samples treated with 0.3mM aluminum, copper, iron, or zinc. Only the samples treated with zinc displayed a loss of octreotide The 500ng/mL concentration of octreotide is converted to micromoles (µm) and compared to the 300 µm concentration of the metals to show the ratio between octreotide and the metals ng/ml octreotide in 20:80 acetonitrile/water with 0.1% formic acid samples treated with aluminum, copper, iron, or zinc after being stored in a refrigerator for 13 days Masses of the amino acids and metals involved in the research...59 v

6 LIST OF FIGURES Table Page 1. Structure of Octreotide Structure of Somatostatin Picture of unhemolyzed matrix and three different levels of hemolysis Structure of Heme Structure of Artemisinin A diagram of a standard solid phase extraction cartridge An example of solid phase extraction Examples of analytical, narrow bore, and capillary HPLC columns The structure for the steroids Estradiol and Equilin-d Schematics of 4 basic pore types used in HPLC stationary phase A representation of an injector valve in the load, where the sample is place inside the loop. When the valve switches to the inject position the sample is washed off of the loop into the HPLC liquid stream An example of a basic HPLC setup A representation of a single quadrupole mass spectrometer Quadrupole images, the left side image is looking down the barrel of the quadrupoles and the right image is a side view of ion oscillating down the quadrupoles The schematic shows a simplified view of the LC/MS/MS analysis of a sample A display of a mass spectrometry scan where the intensity is displayed on the y-axis as counts per second (cps), and the mass-to-charge ratio (m/z) is displayed on the x-axis A total ion HPLC-MS chromatogram where the intensity is displayed on the y-axis as counts per second (cps), and the time is displayed on the x-axis in minutes Octreotide in plasma treated with hemolyzed whole blood at 3 different incubation times...30 vi

7 19. Octreotide internal standard in plasma treated with hemolyzed whole blood at 3 different incubation times Octreotide in plasma treated with non-hemolyzed whole blood at 3 different incubation times Octreotide internal standard in plasma treated with non-hemolyzed whole blood at 3 different incubation times Q1 scan of the 500ng/mL octreotide control sample in 20:80 acetonitrile/water with 0.1% formic acid Q1 scan of a 500ng/mL octreotide sample in 20:80 acetonitrile/water with 0.1% formic acid treated with aluminum Q1 scan of a 500ng/mL octreotide sample in 20:80 acetonitrile/water with 0.1% formic acid treated with copper Q1 scan of a 500ng/mL octreotide sample in 20:80 acetonitrile/water with 0.1% formic acid treated with iron Q1 scan of a 500ng/mL octreotide sample in 20:80 acetonitrile/water with 0.1% formic acid treated with zinc High pressure liquid chromatography analysis of the 500ng/mL octreotide control sample in 20:80 acetonitrile/water with 0.1% formic acid High pressure liquid chromatography analysis of a 500ng/mL octreotide sample in 20:80 acetonitrile/water with 0.1% formic acid treated with aluminum High pressure liquid chromatography analysis of a 500ng/mL octreotide sample in 20:80 acetonitrile/water with 0.1% formic acid treated with iron High pressure liquid chromatography analysis of a 500ng/mL octreotide sample in 20:80 acetonitrile/water with 0.1% formic acid treated with zinc A scan of mass 410 to 620 from the time point of 1 to 1.6 minutes for the octreotide control sample A scan of mass 410 to 620 from the time point of 1 to 1.6 minutes for octreotide treated with iron A scan of mass 410 to 620 from the time point of 1 to 1.6 minutes for octreotide treated with zinc A scan of mass 410 to 620 from the time point of 1 to 1.6 minutes for octreotide treated with aluminum...53 vii

8 35. A full scan from the time point of 0.85 to 1.1 minutes of the HPLC analysis of octreotide treated with aluminum after a thirteen day incubation A full scan from the time point of 0.85 to 1.1 minutes of the HPLC analysis of octreotide treated with iron after a thirteen day incubation A full scan from the time point of 0.85 to 1.1 minutes of the HPLC analysis of octreotide treated with zinc after a thirteen day incubation Full scan from the time points of 0.86 to 1.22 minutes of the HPLC analysis of the octreotide control sample after a thirteen day incubation...58 viii

9 INTRODUCTION Octreotide, H-D-Phe-Cyc-Phe-D-Trp-Lys-Thr-Cyc-Thr-ol, Figure 1, [1] is a synthetic analog of the octapeptide hormone somatostatin [2] which is a growth hormone-inhibiting hormone (GHIH). Somatostatin, Figure 2, regulates the endocrine system and affects neurotransmission and cell proliferation by interacting with G-protein-coupled somatostatin receptors and is involved in the release and inhibition of many secondary hormones. Octreotide is a more potent inhibitor of insulin, growth hormones and glucagon than the naturally occurring somatostatin. The salt form of octreotide, octreotide acetate, is the FDA approved form of this peptide and is what is available commercially to patients. Figure 1: Octreotide [3]

10 Figure 2: Somatostatin [4] During clinical trials, blood specimens are drawn from patients at several time intervals after the drug has been administered. The data from this analysis can then be used to determine how quickly a drug enters the bloodstream and how long it takes the body to metabolize the drug and remove it from the bloodstream. Once the specimens are drawn, they are centrifuged to separate the red blood cells, platelets, serum and the plasma layer. Samples from the plasma layer were used in this research. Sometimes when specimens are drawn they are mishandled and the red blood cells are damaged prior to being centrifuged causing their cellular components to leak out into the rest of the collected specimen. This process is called hemolysis [5] and some of the causes in specimen mishandling are: blood being drawn too vigorously into a syringe, the needle diameter is too restrictive for blood flow, or the specimen is shaken or vortexed too vigorously prior to centrifugation. As shown in Figure 3, moderate or greater hemolysis can be 2

11 easily confirmed if the normally yellow colored plasma specimen has a pink or even reddish hue, but even minor amounts of hemolysis that are not visible to the unaided eye will result in trace increases in enzymes levels in samples. [6] Figure 3: The left tube is an unhemolyzed specimen and the tubes on the right display an increasingly higher percentage of hemolysis. [7] This research was done to see if the peptide octreotide is affected by the presence of hemolyzed matrix and in particular by the presence of heme, a component of hemoglobin. Hemoglobin comprises about 35% of the total volume of the components within a red blood cell. It is comprised of four globular protein subunits; each comprised of a protein chain tightly bound to a non-protein heme molecule. Heme, Figure 4, is comprised of a single iron ion, Fe (II), at the center of an organic heterocyclic ring called a porphyrin. 3

12 Figure 4: Heme [8] When a cell membrane is ruptured, its contents leak out into the rest of the specimen and can cause one of three issues: 1. The extra material escaping from the ruptured cells can change the overall concentration of the sample by dilution [9]. If the compound of interest is found in higher concentrations in plasma, then when the cell membrane is ruptured the sample will become diluted and the analytical results will be lower. If the compound is found inside the blood cells then the analytical results may be higher because when the cell membrane is ruptured more of the drug will be available for analysis. 2. The released material from hemolysis can increase the absorbance in the short wavelength range. Analytical results that rely on photometric measurements may have an increase in the detected signal leading to erroneous data. 3. Some chemical reactions can be affected by the presence of the hemoglobin and in other cases the actual chemical tests can be effected [10]. 4

13 In a paper by Zhang and Gerhard [11], the authors noted that heme reduced the endoperoxide bridge within artemisinin, Fig 5, and caused the activation of the drug. Heme (Fe 2+ protoporphyrin-ix) was more efficient in the reduction than inorganic iron or hemoglobin. Because sulfur is a VIA element like oxygen and forms double bonds and hydrogen bonding in the same manner, the reduction observed with the endoperoxide bridge could be possible with disulfide bonds in other compounds. This research was designed to see if the disulfide bond in octreotide would be affected like artemisinin s endoperoxide bridge. Figure 5: Artemisinin [12] 5

14 METHODS Octreotide acetate was procured from Sequoia Research Products and the internal standard (IS) octreotide [ 13 C 6 Phe3] was procured from Pharmaceutical Product Development. HPLC grade methanol and acetonitrile were purchased from Burdick and Jackson. Ammonium hydroxide (30%) and potassium hydroxide (86%) were purchased from J.T. Baker. Formic acid and phosphoric acid were purchased from Mallinckrodt. The reagent water used was filtered by the in house Millipore system. The Isolute mg C18 (EC) SPE plate was purchased from Varian. An Acquity UPLC BEH C18 vanguard pre-column, 2.1mm x 5mm, 1.7 µm, product number and an Acquity UPLC BEH C18 vanguard pre-column, 2.1mm x 100mm, 1.7 µm (product number ) were purchased from Waters. Iron powder (product number 44890), zinc powder (product number 14409), aluminum powder (product number ), iron (II) chloride tetrahydrate (product number ), iron (III) chloride (product number ), hemin (product number 51280), sodium dithionite (product number), and protoporphyrin-ix (product number) were purchased from Sigma-Aldrich. The copper wire (product number 31284) was purchased from Riedel-deHaën. The HPLC equipment used was an Agilent 1200 series binary pump, a Shimadzu LC- 10AD VP series pump, and a 10 port cheminert 2 position selector valve from Valco Instruments Company Incorporated, a hot sleeve-15l set at 50 C from Analytical Sales and Services and an API 4000 series LC/MS/MS mass spectrometer from AB Sciex. The solid phase extraction was automated using a Tomtec Quadra 96 Model 320 liquid-handling system. The stock solutions were prepared at 100 µg/ml in 0.1:20:80 formic acid/acetonitrile/water. The hemolyzed blood was made by taking the K2 EDTA whole blood 6

15 and freezing, thawing, vortexing, and then refreezing for three cycles to ensure that all of the cell membranes had ruptured [7]. During the course of this research octreotide samples were prepared in matrix, human K2 EDTA plasma, and in other tests they were prepared in solution, 0.1:20:80 formic acid/acetonitrile/water (v,v,v). The samples in matrix were prepared for mass spectrometer analysis by separating the analyte of interest, octreotide, from the other components found in the matrix. This separation was done by solid phase extraction [13], SPE, which is a process where compounds suspended in a liquid are separated from the other compounds depending on their chemical or physical properties. Each sample is extracted through an individual cartridge, Figure 6, which is made up of the reservoir, frits, sorbent bed, and luer tip. Depending on the desired action a sorbent bed can be chosen to retain the analyte of interest and allow the unwanted compounds to flow through, or to retain the unwanted compounds and allow the analyte of interest to flow through the sorbent bed. In the case of this research, the chosen sorbent bed of the cartridge, weak cation exchange, retained octreotide and allowed cleaning and then elution of octreotide into a clean reservoir for high pressure liquid chromatography, HPLC, analysis. Figure 6: A diagram of a standard solid phase extraction cartridge. [14] 7

16 There are three main types of sorbent beds [15], or stationary phases, to choose for compound isolation and they are called: normal phase, reverse phase and ion exchange. Ion exchange solid phase extraction is used for analytes that are charged when in solution and is divided into four sub categories of stationary phase: strong anion, weak anion, strong cation and weak cation. A weak cation exchange cartridge was the solid phase extraction type used for this research. Weak cation exchange (WCX) stationary phase is comprised of a carboxylic acid group that is bonded to a silica surface and has a pka of approximately 4.8 so it will be negatively charged in a solution of at least 2 ph units above this pka. The elution solvent for a weak cation exchange stationary phase used to release the analyte from the stationary phase is at least 2 ph units above the weak cation exchange pka value. Strong cation exchange (SCX) stationary phase is comprised of an aliphatic sulfonic acid group that is bonded to a silica surface and has a pka of less than 1. These are used to isolate strong cationic, pka 14 or greater, or a weak cationic, pka of less than 12, compounds in matrix with a ph of a least 2 unit below the analyte s pka. Elution from a strong cation exchange stationary phase is done with a solution that has a ph of at least 2 units higher than the analytes pka. Strong anion exchange (SAX) stationary phase is comprised of an aliphatic quaternary amine group that is bonded to a silica surface and has a pka greater than 14 so that it is charged at all phs when in an aqueous solution. It is used to isolate weak anionic and strong ionic compounds, and a solution with ph 2 units greater than the pka is used as a matrix. Analytes bound to these types of stationary phases are released by a solution at least 2 ph units below the analytes pka. Weak anion exchange (WAX) stationary phase is comprised of an aliphatic 8

17 aminopropyl group that has a pka around 9.8. A solution of at least 2 ph units lower than the pka of the stationary phase is used, but the solution must also be 2 ph units above the analyte s pka. Weak anion exchange is used to isolate strong and weak anions because they can be eluted from the amine functional group by using a solution at least 2 ph units above the stationary phase s pka. Normal phase SPE uses a polar stationary phase to isolate a polar analyte suspended in a mid to nonpolar matrix, like hexane or acetone. The retention mechanism is the interaction between the polar groups of the analyte and the polar functional groups of the sorbent bed. The analyte is eluted from the stationary phase by passing a solvent more polar than the original matrix through the stationary phase. Reverse phase SPE uses a nonpolar stationary phase to bind a mid to nonpolar analyte suspended in a moderately polar or greater matrix. The retention mechanism is the interaction between the carbon-hydrogen bonds of the analyte and the stationary phase. A nonpolar solvent is used to elute the analyte from the stationary phase. A typical solid phase extraction has four steps: conditioning, sample loading, washing and elution, Figure 7. In the conditioning step the dry sorbent bed is treated by adding a solvent, typically methanol, to wet the bed and penetrate the bonded phase within the sorbent bed. Then either water, or a buffer close the composition of the sample is washed through the bed to prepare the bonded phase. In the sample loading step, the sample is added to the reservoir and allowed to slowly pass through the sorbent bed so that the analyte can interact with the material and be retained. During this and all other steps the fluids are allowed to either pass through the bed using gravity, centrifugation or by adding negative pressure to the cartridge using a vacuum pump. The vacuum pump method was used for this research. 9

18 The washing step is done to remove impurities and other interfering material from the sorbent bed without causing the analyte to be released. This washing step is typically done with water or an organic compound that will not disrupt the interaction between the analyte and the sorbent bed. Once washed the cartridge is moved so that the analyte may be eluted into a clean container and the analyte is then washed from the cartridge with a solution that severs the interaction between the analyte and the sorbent bed. Sometimes the elution solvent is not compatible with the chosen HPLC column and the elution solvent must be removed, typically by evaporation, and the dried sample reconstituted with a solvent more compatible for HPLC analysis. Figure 7: An example of solid phase extraction. The conditioning step wets the stationary phase and prepares it for the loading phase where the analyte binds to the stationary phase. The washing step removes impurities that might interfere with analysis after the analyte is removed from the stationary phase during the elution step. [14] High pressure liquid chromatography [16], HPLC, is a form of column chromatography where a solvent is forced under high pressures of up to 6000 psi through a column that has been 10

19 packed with a stationary phase. By using higher pressures, smaller particle sizes can be used in the stationary phase allowing for more interaction between the stationary phase and the compounds suspended in a sample. The use of high pressure also allows for quicker elution times and the compounds can be eluted in a much narrower band, increasing the sensitivity of the assay. There are several variants of chromatography available to separate analyte(s) from other components within a sample following sample introduction. The chromatography variants are normal phase, reverse phase, ion exchange, displacement, size-exclusion, and bioaffinity. The choice of solvent, or mobile phase, and the packing material of the column determine what variant of chromatography that is being used. The packing material and mechanisms of isolation and elution for normal phase, reverse phase and ion exchange are similar to those used in solid phase extraction. Figure 8: Examples of analytical, narrow bore, and capillary HPLC columns. [17] The other factors in high pressure liquid chromatography columns are the internal diameter, length, particle size, pore size, temperature, and the amount of pressure applied to the 11

20 mobile phase. There are four categories of internal diameters, Figure 8, for high pressure liquid chromatography columns: large, analytical, narrow-bore, and capillary. Large ID columns are over 10 mm in diameter and are used for industrial applications. Analytical scale columns have an internal diameter of 4.6 to 6 mm and narrow-bore columns average 1 to 2 mm in internal diameter and are used for fluorescence and liquid chromatography-mass spectrometry analysis. Capillary columns which are under 0.3 mm in diameter are made from fused silica capillaries instead of stainless steel that is used in the other columns. Column length is another factor when choosing a high pressure liquid chromatography column because the extra amount of stationary phase allows for more interaction with the analyte(s) being isolated. When dealing with a sample that has molecularly similar analytes the extra amount of stationary phase can be critical. The steroids estradiol and the deuterated equilin internal standard (equilin-d 4 ), Figure 9, are good examples of this situation because they have the same mass and the only differences between these two are that estradiol has an alcohol in the 17 position while equilin-d 4 has a ketone at the 17 position, a double bond between the carbons at the 7 and 8 position, and deuterium instead of hydrogen at four locations. Some assays require the monitoring of the steroids estrone, estradiol, and equilin along with their deuterated internal standards and this is when separation can become a critical. Separation of these compounds requires high efficiency chromatography afforded by increased column length and/or reduced particle size. 12

21 Figure 9: The structure for the steroids Estradiol and Equilin-d. [18] The particle size of a column refers to the size of the silica beads that make up the stationary phase. The smaller the particle size the greater the total surface area inside the column which can improve separation, but the smaller particle size requires a greater amount of mobile phase pressure to maintain good analyte separation across the stationary phase. Particle sizes vary depending on the application the column is being used for, but 3 µm and 5 µm are the most commonly used particles sizes. Columns with a particle size below 3 µm are used in ultra performance liquid chromatography, UPLC. Columns and the pressure required for optimum performance can reach up to psi in UPLC. Many of the stationary phases used in high pressure liquid chromatography analysis are porous [19], Figure 10, which means that the stationary phase has little pockets in their structure, and these pores increase the available surface area inside the column. This increased surface area allows for more interaction between the stationary phase and the analytes allowing for greater separation. 13

22 Figure 10: Schematics of 4 basic pore types used in HPLC stationary phase; (a) totally porous, (b) perfusion, (c) nonporous, and (d) superficially porous particles. [19] The consistency of the pressure applied to the mobile phase by a pump is critical for the reproducibility of a high pressure liquid chromatography assay. Many high pressure liquid chromatography methods use more than one solution as their mobile phase and these solutions are applied by a pump and mixed prior to entering the column. The mixture is changed slowly by a gradient process to release the analyte from the stationary phase and allow the analyte to flow downstream to the detector. The efficiency of a high pressure liquid chromatography column can be improved by raising its temperature [20] because at elevated temperatures the viscosity of liquids decrease and the diffusion coefficient increases. The increased diffusion coefficient allows for better interaction between the stationary phase and the analyte and can improve peak shape. This also reduces the backpressure caused by the tightly packed stationary phase of the column which decreases the stress on the HPLC pump, improving its efficiency. 14

23 Figure 11: A representation of an injector valve in the load, where the sample is place inside the loop. When the valve switches to the inject position the sample is washed off of the loop into the HPLC liquid stream. [21] Using reverse-phase chromatography, the form used for this research project, the initial composition of the mobile phase can be, for example, a mostly aqueous moderately polar solvent that closely resembles the eluent from the solid phase extraction used to isolate the analyte from the matrix. The sample is applied to the mobile phase stream by an injector, Figure 11, which is a valve that allows the user to insert an aliquot of the sample into a section of tubing called a loop when the injector is in the load position. The valve is actuated and the injector switches to the inject position that moves the loop in line with the pump, Figure 12, and flushes the sample downstream into the high pressure liquid chromatography column. Once the sample reaches the column, the analyte interacts with the stationary phase and is held in place by van der Waals forces of the carbon-hydrogen interaction. The composition of the mobile phase is slowly changed to flush away unwanted material that has some affinity for the stationary phase, and then the mobile phase reaches a sufficient nonpolar composition that the analyte is washed out of the column and into the detector. 15

24 Figure 12: An example of a basic HPLC setup. [21] The detector used was a quadrupole mass analyzer mass spectrometer [22] which is an analytical instrument used to measure the mass-to-charge ratio (m/z) of charged particles. The basic principle is to introduce a compound, which in case of the research is dissolved in a liquid mobile phase, and ionize the chemical compounds to generate charged molecules and then measure their mass-to-charge ratio. Once a sample is loaded into a mass spectrometer it undergoes vaporization and the components are ionized. There are several forms of ionization coupled to mass spectrometers, such as: electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), fast atom bombardment (FAB), photoionization (PI), field desorption (FD), direct analysis in real time, and secondary ion mass spectrometry. The one used in the course of this research was electrospray ionization [23]. The ions are separated according to their mass-to-charge ratio by an electromagnetic field generated by four circular rods, called quadrupoles, which are set in parallel to each other, Figure 16

25 13. The separation is done by stabilizing the trajectory of the selected mass-to-charge ratio in the oscillating field generated by the quadrupoles, Figure 14. The opposing rods of the quadrupoles are connected together electrically and a radio frequency is applied to each pair of the rods. Ions that do not have the correct mass-to-charge ratio have an unstable trajectory and ultimately collide with one of the rods and are deflected away from the detector located at the end of the quadrupoles. The electrical potentials of the quadrupoles can be changed to allow a wide range of mass-to-charge values to be monitored in either large groups or in succession. Figure 13: A representation of a single quadrupole mass spectrometer. [24] 17

26 Figure 14: Quadrupole images, the left side image is looking down the barrel of the quadrupoles and the right image is a side view of ion oscillating down the quadrupoles. [25] The Sciex 4000 series mass spectrometer used for this research is a type of mass spectrometer called a tandem mass spectrometer, or LC/MS/MS. A tandem mass spectrometer [26], Figure 15, is capable of two rounds of mass spec isolation of particles separated by molecular fragmentation. Three quadrupoles are aligned in a series and in the first quadrupole (Q1) the analytes from the ion source are isolated prior to entering the second set of quadrupoles. The second set of quadrupoles stabilizes the ions while they collide with a gas, in this case nitrogen, causing them to fragment by collision-induced-dissociation (CID). In the third quadrupole (Q3) the resulting fragments are sorted and the radio frequency is optimized so a specific mass-to-charge ratio will reach the detector located just beyond the third quadrupole. This allows for greater selectivity from a sample that may contain multiple compounds with the 18

27 same mass-to-charge ratio, but due to their structure they may produce different fragments. Tandem mass spectrometry allows for much greater selectivity than the single quadrupole. Also, researchers can deduce the structure of the initial compound by studying the fragments produced in the collision cell. This is done by isolating the known compound mass in Q1 and after fragmentation in the collision cell (Q2), Q3 is set to scan from the mass-to-charge ratio of the parent compound and lower so that all the produced fragments can be found. The size and intensities of these fragments can be then used to deduce the structure of the original compound. Figure 15: The schematic shows a simplified view of the LC/MS/MS analysis of a sample. At the end of the quadrupoles is a detector that records either the charge induced or the current produced when an ion passes through or impacts the surface. These ions pass through an electron multiplier called a Faraday cup to increase the signal received from the quadrupoles because the amount of ions can be quite small. Once collected the signal response from the detector can be displayed as intensity, or counts per second (cps), on the y-axis versus the massto-charge ratio (m/z), on the x-axis, Figure 16. This type of spectrum only displays the total amount of material with a specific mass-to-charge ratio in a sample, but cannot distinguish 19

28 multiple compounds of that ratio in the sample. Usually these types of scans do not involve liquid chromatography prior to introduction into the mass spectrometer, but are done by a direct infusion of a sample into the ion source. Figure 16: A display of a mass spectrometry scan where the intensity is displayed on the y-axis as counts per second (cps), and the mass-to-charge ratio (m/z) is displayed on the x-axis. 20

29 Figure 17: A total ion HPLC-MS chromatogram where the intensity is displayed on the y-axis as counts per second (cps), and the time is displayed on the x-axis in minutes. Figure 17 shows a display of a chromatogram in which high pressure liquid chromatography separation is done to the sample prior to entering the ion source of the mass spectrometer. The mass spectrometer is set to scan a specific mass or range of masses being introduced into the ion source. The chromatogram displays the intensity (counts per second) of what is scanned versus the duration of the scan. This allows for compounds that have the same mass-to-charge ratio to be separated prior to entering the mass spectrometer and gives confidence that the data represents the compound(s) intended for analysis. 21

30 Samples were analyzed using a procedure validated by PPD [27]. Into a 96-position, 2.0-mL, square-well, conical bottom, polypropylene plate, a 200 µl aliquot of sample in K2 EDTA human plasma was added and then into each well and 500 µl of phosphate buffer, ph 7.0 was added. Using a Tomtec automated solid phase extraction pipetting system an Isolute mg C18 (EC) SPE plate was conditioned three times with 300 µl of methanol, followed by 300 µl of reagent water, also administered three times. After each addition vacuum was applied carefully so that the bed material did not completely dry out. The sample was then loaded onto the SPE plate by transferring three 275 µl aliquots, and between each transfer a partial vacuum was applied followed briefly by full vacuum. The SPE plate was then washed three times with 300 µl of water, followed by three 200 µl treatments with 30:70 acetonitrile/water. A full vacuum was applied with each addition. Next the sample was eluted from the SPE plate with three 300 µl additions of 2:8:90 by volume 29% ammonium hydroxide/water/methanol into a new 96-position, 2.0-mL, square-well, conical bottom, polypropylene plate that was placed in the vacuum chamber. The eluent was evaporated under a nitrogen stream at 45⁰C, and once dry the samples were reconstituted with 150 µl of 1:19:80 ammonium hydroxide/acetonitrile/water. The HPLC separation was performed with an Xterra MS C18, 2.1 mm ID and 50 mm length column using 0.5% ammonium hydroxide as mobile phase A and acetonitrile as mobile phase B; see Table 1 for the chromatographic gradient conditions. The column was attached to a Sciex 4000 series LC/MS/MS mass spectrometer set to positive mode electrospray ionization. Mobile phase A, 0.5% ammonium hydroxide in water, and mobile phase B, acetonitrile, were used for the chromatographic gradient. 22

31 Total Time (min) Flow Rate (µl/min) Mobile Phase A (%) Mobile Phase B (%) Step Table 1: The high pressure liquid chromatography gradient program used for the analysis of octreotide samples. A second extraction procedure was used in the later experiments due to concerns with the stability of the 2:8:90 ammonium hydroxide/water/methanol used as the elution solution for the C18 SPE plate. Another chemist in the lab reported that she had noticed a difference in recovery between SPE plates in a multi plate extraction session, and after some investigation it was determined that the 2:8:90 ammonium hydroxide/water/methanol mixture could not be older than a few hours between plate extractions or the amount recovered from the SPE plate would be lower in the second plate. To eliminate possible bias when comparing different sample extractions to each other, a newer extraction method that uses a WCX SPE plate with a 1% trifluoroacetic acid (TFA) in 75:25 acetonitrile/water as the elution solution was used [28]. Into a 96-position, 2.0 ml, square-well, conical bottom, polypropylene plate, a 200 µl aliquot of sample was added into each well, followed by 500 µl of phosphate buffer, ph 7.0 and 50 µl of 30 ng/ml internal standard working solution. Using a Tomtec automated extraction solid phase extraction pipetting system, a WCX µelution SPE plate was conditioned with 200µL of methanol, followed by 200 µl of reagent water. After each addition the vacuum was applied, being careful not allow the bed material to completely dry out. The sample was then loaded onto 23

32 the SPE plate by transferring two 225 µl aliquots. Between each transfer a partial vacuum was applied then the full vacuum was applied briefly. The SPE plate was then washed twice with 200 µl with 5% ammonium hydroxide, and then twice with 200 µl with 20:80 acetonitrile/water. A full vacuum was applied after each addition. Next the sample was eluted from the SPE plate with 25 µl 1% TFA in 75:25 acetonitrile/water two times into a new 96-position, 2.0 ml, square-well, conical bottom, polypropylene plate that was placed in the vacuum chamber. The eluent was diluted with 150 µl of water through the plate and the plate removed and the elution block was sealed and vortexed for 1 minute. The HPLC separation was performed with an Acquity UPLC BEH C18, 2.1mm x 100mm, 1.7 µm pore size, and the column was heated to 50 C to improve efficiency. See Table 2 for the gradient program. Mobile phase A, 0.1% formic acid in water, and mobile phase B, 0.1% formic acid in acetonitrile, were used for the chromatographic gradient. The flow from the column was attached to a Sciex 4000 series LC/MS/MS mass spectrometer set to positive mode electrospray ionization. Step Total Time (min) Flow Rate (µl/min) Mobile Phase A (%) Mobile Phase B (%) Table 2: The high pressure liquid chromatography gradient program used for the second octreotide sample analysis method. Samples were prepared at different concentrations of hemolyzed whole blood in plasma. The control sample was 0% hemolyzed whole blood in plasma and had only octreotide and the 24

33 octreotide internal standard added. The 2% hemolyzed whole blood in plasma was used as an example of mild hemolysis of a specimen and 20% hemolyzed whole blood in plasma was prepared to represent the extremely hemolyzed sample. An 8% hemolyzed whole blood in plasma was prepared to give a third data point for trending. These were compared to samples with non-hemolyzed whole blood at the same concentrations. Most of the experiments were done at two different temperature settings to see if room temperature or 37 C, average human body temperature, would enhance or suppress any effects hemolyzed blood would have on octreotide. Room temperature was chosen because most samples are not immediately centrifuged to separate the plasma from the rest of the material in whole blood and body temperature was used because most causes of hemolysis happen at the time of collection and the specimens would still be warm when any effects would be caused by the hemolyzed red blood cells. These samples at room temperature and 37 C were also subdivided into incubation times to see if a 0 hour, or no incubation time, at these temperatures would generate a different response than an incubation time of 3 hours. Reimers, McCann, Cowan and Concannon [29] showed that peptides like insulin, which has disulfide bonds in its structure, were affected by storage time, hemolysis and storage temperature but they only reported results at room temperature and 4 C. Another set of samples was prepared to test whether adding octreotide to the sample prior to the introduction of hemolyzed matrix would have an effect on recovery. This was done to see if octreotide had become protein bound in the plasma layer, protecting it from the effects of the hemolyzed blood. Four sets of samples were prepared; 1) octreotide was added into a plasma sample and then diluted fourfold with additional plasma, 2) octreotide was added into a plasma sample diluted fourfold with hemolyzed whole blood, 3) octreotide was added to a mixture of 25

34 25% hemolyzed whole blood in plasma and the sample was diluted with additional 25% hemolyzed whole blood in plasma, 4) octreotide was added into hemolyzed whole blood which was then diluted fourfold with plasma. The prepared samples were allowed to equilibrate for thirty minutes to being diluted. The final extraction test was conducted by preparing a high concentration sample of octreotide at 1000ng/mL in three different matrixes, K2 EDTA human plasma, 20% hemolyzed whole blood in K2 EDTA human plasma, and 5% hemolyzed whole blood in K2 EDTA human plasma. These were allowed to equilibrate for 1 hour at room temperature before a ml aliquot was taken and diluted to 1mL, a 10 fold dilution with K2 EDTA human plasma, which was allowed to equilibrate at room temperature for 30 minutes. This sample was diluted again 10 fold with K2 EDTA human plasma and allowed to equilibrate for 30 minutes before aliquots were taken and extracted in six replicates for the experiment. Test samples of 10 ng/ml octreotide were prepared in K2 EDTA human plasma. Spiking solutions of hemin were prepared at 2 mm and 4 mm in 0.05 N sodium hydroxide, also a spiking solution of 4 mm sodium dithionite was prepared in 0.05 N sodium hydroxide. The sodium hydroxide used for the hemin and sodium dithionite were degassed with helium prior to use and the hemin and sodium dithionite powder was put in a 37 C vacuum oven. After an hour under vacuum the chamber was filled with argon gas and the samples were quickly spiked with sodium hydroxide. All of this was done to remove as much oxygen from the samples so that when the sodium dithionite is added to the hemin it will reduce the hemin protein from an oxidized form creating heme. Removing as much oxygen as possible from the sodium hydroxide prior to preparing the sodium dithionite solution helped to ensure that the hemin was converted to heme. Heme contains Fe 2+ protoporphyrin-ix and it should be reactive to the disulfide bonds in 26

35 octreotide whereas the Fe 3+ protoporphyrin-ix of hemin is in the highest oxidation for iron and is not reactive. Also prepared were solutions of protoporphyrin IX (1.13 mg/ml), ferric chloride (397.6 mg/ml) and ferrous chloride (324.4 mg/ml), each of these solutions were prepared at a concentration 2mM in water. The amount of free iron found in a highly hemolyzed sample prior to the sample being centrifuged is 860 ng/ml [6]. Because iron settles in the serum phase after centrifugation, the amount of free iron in plasma after centrifugation is almost negligible except in cases of rare disorders. A 1 ml aliquot of the 4 mm heme and a 1 ml of 4mM sodium dithionite were mixed so that the sodium dithionite would convert the hemin to heme and make a 2mM (1.23 mg/ml) solution [30]. The concentration of 1.23 mg/ml is equivalent to 1% hemolyzed blood in plasma. Six 4mL aliquots of the 10ng/mL octreotide were taken from the pool and each was treated with one of the prepared solutions; heme, hemin, sodium dithionite, protoporphyrin IX, ferric chloride and ferrous chloride. These solutions were allowed to equilibrate for 1 hour at 37 C before aliquots were taken and extracted along with a control of 10 ng/ml octreotide in K2 EDTA plasma. In an early experiment a stock solution of octreotide was found to have degraded within two weeks of being prepared possibly because of the use of an aluminum weigh boat that was left in the solution at the time of preparation. Upon investigation the 100 µg/ml octreotide stock solution, which had been fresh in the first experiment, had degraded to such an extent that the peak response was tenfold less than a freshly prepared stock. The aluminum weigh boat may have caused a metal ion catalyzed reduction of octreotide. Erlandsson and Hällbrink (31) observed that zinc broke the disulfide bonds in certain peptides and aluminum may be having a similar effect on octreotide. A new stock solution, prepared using a glass weigh boat, was 27

36 compared to the solution that had been prepared with an aluminum weigh boat to confirm the loss of octreotide and the new stock was used in the future experiments. A test was done to determine if zinc II, aluminum III, copper I, copper II, or iron III would have an effect on the recovery of octreotide in solution. A 0.49 µm intermediate solution of octreotide in 20:80 acetonitrile/water with 0.1% formic acid was prepared and 2mL of the intermediate was aliquoted into10ml vials. A 300 µm solution of each metal was prepared and added to a vial. A second set of vials was prepared and to them was added 10uL of methyl acrylate (MA), which will bind to any broken disulfide bonds preventing them from reforming. Copper was chosen as a control metal since it has a positive standard electrode potential, Table 3, and should not react with octreotide. Table 3: The standard electrode potentials of aluminum, copper iron and zinc. The solutions were vortexed and allowed to sit at room temperature for 1 hour before being centrifuged at 4000rpm for 15 minutes. The samples were then infused directly into the mass spectrometer and a Q1 scan was done with a scan window from 410 to 610 m/z, see figures 23-27, to see if there was any response loss at the 510 mass. Octreotide s molecular mass is g/mol, but it is a dual charged molecule so the m/z (mass/charge) ratio is approximately 510 m/z. A ml aliquot was placed into a 96 well block and a 5uL injection was made and the sample was separated by HPLC using an Acquity UPLC BEH C18, 1.7 µm pore size,

37 column inner diameter, and 100 cm long column. This was done to help chromatographically separate degraded material resulting from the interaction of the metals with octreotide. The high pressure liquid chromatography eluent was analyzed by a Q1 scan of the mass-to-charge ratio range of 410 to 610 m/z. The solutions were stored at 2-8 C and were assayed again 13 days later to see if there was any further degradation of octreotide caused by the presence of metal in the solution. The scan window was narrowed to scan between the range of 450 to 560 m/z, see figures

38 RESULTS AND DISCUSSION Hemolyzed whole blood was added to plasma containing octreotide at different percentages and another set of samples were created using non- hemolyzed matrix. The initial results of comparing 0, 2, 8, and 20 % hemolyzed whole blood to the same percentages in nonhemolyzed whole blood was promising because the samples with hemolyzed matrix showed a definite trend in recovery loss as the percentage of hemolyzed matrix was increased, Figure 18 (octreotide), and Figure 19 (internal standard). The samples that had the same concentrations of unhemolyzed whole blood did not show the same trend, Figure 20 (octreotide) and Figure 21 (internal standard). The incubation samples had nearly the same response in the hemolyzed and non-hemolyzed samples except for the 0 % hemolyzed at the 3 hour incubation time which was much higher and was determined to be a double spike of the spiking solution. The actual cause of the recovery loss was not due to the degradation of octreotide by hemolyzed whole blood, but likely due to the extra cellular material clogging the pores of the solid phase bed of the extractions plates. Figure 18: Octreotide in plasma treated with hemolyzed whole blood at 3 different incubation times. 30

39 Figure 19: Octreotide internal standard in plasma treated with hemolyzed whole blood at 3 different incubation times. Figure 20: Octreotide in plasma treated with non-hemolyzed whole blood at 3 different incubation times. 31

40 Figure 21: Octreotide internal standard in plasma treated with non-hemolyzed whole blood at 3 different incubation times. In the comparison of a fourfold dilution (dil4) of 100 ng/ml octreotide in K2 EDTA human plasma samples that were diluted with hemolyzed blood did have a much lower recovery than the sample prepared and diluted with plasma, Table 4. The results from where octreotide was added to the samples containing hemolyzed whole blood were also lower than the samples prepared completely in plasma, but not as low of a recovery as the plasma samples diluted with hemolyzed blood, see Table 4. The expectation was that the plasma dil4 with hemolyzed and the 25% hemolyzed in plasma dil4 with additional 25% hemolyzed should have had the same amount of recovery. Since the matrix to be added was freshly vortexed prior to addition, the sample diluted with hemolyzed whole blood would have gotten more cellular material than the sample with 25% hemolyzed whole blood in plasma. This additional material would have competed with the binding sites on the solid phase bed more than the other samples. The internal standard tracked very similarly to octreotide and the internal standard was not added until after the 4% phosphoric acid had been added to the sample aliquots. If the hemolyzed matrix was causing degradation of octreotide the reaction should have been stopped or at least reduced by 32

41 the addition of the 4% phosphoric acid, and the four different preparations should have had internal standard responses that were more comparable. This reinforces the hypothesis that the active sites on the stationary phase of the solid phase extraction cartridges were being blocked by the material from the hemolyzed matrix. Plasma diluted with plasma (cps) Plasma diluted with hemolyzed blood (cps) 25%hemolyzed diluted with 25% hemolyzed blood (cps) Hemolyzed blood diluted with plasma (cps) Octreotide Mean Std Dev Octreotide Internal Standard Plasma diluted with plasma (cps) Plasma diluted with hemolyzed blood (cps) 25%hemolyzed diluted with 25% hemolyzed blood (cps) Hemolyzed blood diluted with plasma (cps) Mean Std Dev Table 4: The fourfold dilution of samples containing octreotide and octreotide internal standard in four different matrix combinations. The data point highlighted in red for the internal standard sample of plasma diluted with hemolyzed blood was eliminated from the calculations. 33