CN116547489A - Method for drying biocompatible SPME coating - Google Patents

Abstract

An improved process for drying biocompatible solid phase microextraction (BioSPME) coatings results in coatings having highly consistent extraction efficiencies and biocompatibility. A method suitable for a flow-through drying system includes determining a relative humidity in and around the drying system, determining a drying temperature range based on the relative humidity, and maintaining the drying temperature within the determined range while drying the coating.

Description

Method for drying biocompatible SPME coating

Cross Reference to Related Applications

The present application claims priority from U.S. provisional patent application Ser. No. 63/121,071, filed on 3 months of 12 in 2020, 63/121,050, filed on 3 months of 12 in 2020, and 63/121,035, filed on 3 months of 12 in 2020, each of which is incorporated herein by reference in its entirety.

Background

Solid Phase Microextraction (SPME) is a rapid, economical and versatile sample preparation technique. SPME involves extracting the analyte onto a small volume of coating on a substrate (e.g., fiber) and then desorbing the analyte in a gas chromatography syringe or in an organic solvent for liquid chromatography separation and detection. The coating on the SPME substrate is the core of the device. The SPME coating includes a polymer binder and a solid adsorbent. For Liquid Chromatography (LC) applications, it is desirable that the SPME coating is biocompatible to minimize interference from the sample matrix. Some particularly useful biocompatible SPME coatings are composed of functionalized silica (e.g., C18 (octadecyl functionalized) silica) in Polyacrylonitrile (PAN). It is typically coated onto fibers that are commercially available as SPME LC probe products.

The PAN/C18 coating was prepared as follows: PAN is dissolved in a solvent (e.g., DMF), the solution is mixed with C18 silica to form a slurry, the PAN/C18 slurry is coated on a substrate, and the solvent is evaporated, which includes drying the coating at an elevated temperature. The drying conditions disclosed by museata et al (anal. Chem.2007,79,6903-6911) are 1.5 minutes at 180 ℃, but there is no provision for controlling humidity during the drying process. Conventional drying methods follow this procedure. However, it has been found that this approach may lead to inconsistent results, such as differences in morphology, efficacy and biocompatibility of the coating.

There is a need for a new, controlled method of drying biocompatible SPME coatings that results in consistent morphology, efficacy and biocompatibility.

Disclosure of Invention

A new method for drying Solid Phase Microextraction (SPME) coatings, particularly biocompatible SPME coatings that produce BioSPME devices with consistent extraction efficiency and biocompatibility, is provided. The method comprises the following steps: determining the relative humidity (RH percentage or RH%) measured at 22±2 ℃ for a drying system comprising a drying gas, such as air or nitrogen, applying a suitable drying temperature range to the drying system based on the relative humidity, introducing means for coating in a flow-through drying system; and maintaining the drying temperature in the drying system within a selected temperature range for a time sufficient to dry the coating. In some embodiments, the drying system is a flow-through drying system.

In a first embodiment, wherein the RH% is greater than 60%, the drying temperature is maintained in the range of 110 ℃ to 160 ℃.

In a second embodiment, wherein the RH% is in the range of 40% to 60%, the drying temperature is maintained in the range of 80 ℃ to 110 ℃.

In a third embodiment, wherein the RH% is in the range of 15% to 40%, the drying temperature is maintained in the range of 60 ℃ to 80 ℃.

In a fourth embodiment, wherein the RH% is less than 15%, the drying temperature is maintained in the range of 10 ℃ to 50 ℃.

The biocompatible coatings provided herein include a binder selected from the group consisting of Polyacrylonitrile (PAN), polyacrylamide, polyethylene glycol (PEG), polypyrrole, derivatized cellulose, polysulfone, polydimethylsiloxane (PDMS), polyacrylate, polytetrafluoroethylene, and polyaniline, and an adsorbent selected from the group consisting of functionalized silica, carbon, polymer resins, and combinations thereof. In some embodiments, the adsorbent is a C18, C8 or mixed mode functionalized silica. In other embodiments, the adsorbent is a resin selected from the group consisting of HLB resins, divinylbenzene resins, styrene resins, and styrene-divinylbenzene copolymer resins.

Drawings

Fig. 1 shows SEM images of PAN/C18 coatings dried at 110 ℃ at different humidity levels. Fig. 1A: the coating was dried at 20% RH; fig. 1B: the coating was dried at 39% RH; fig. 1C: the coating was dried at about 48% RH; and fig. 1D: the coating is dried at about 60-70% RH.

Figure 2 shows SEM images of PAN/C18 coatings dried at 22 ℃ at different humidity levels. Fig. 2A: the coating was dried at 39% RH; fig. 2B: the coating was dried at 27% RH; fig. 2C: the coating was dried at 23% RH; fig. 2D: the coating was dried at 16% RH; fig. 2E: the coating was dried at 10% RH, and fig. 2F: the coating was dried at 7% RH.

Detailed Description

Conventional drying methods for biocompatible SPME coatings such as PAN/C18, for example, as disclosed by Musteaa et al, are methods that expose PAN/C18 slurry to elevated temperatures under ambient atmospheric conditions to evaporate the solvent. However, it has been found that the resulting coatings do not satisfactorily have inconsistent morphology, efficacy and biocompatibility without the clear reasons for these differences.

It has now been unexpectedly found that biocompatible SPME coatings having consistent morphology, efficacy and biocompatibility can be produced by controlling the humidity during the drying process. Even more surprisingly, the inventors have found that by controlling the humidity level during drying, the drying temperature can be reduced while still providing consistent morphology, efficacy and biocompatibility.

In the present invention it was observed that the morphology of biocompatible SPME coatings (e.g. PAN/C18) varied with humidity level during the drying step and that the dried coatings only showed good efficacy and biocompatibility at specific temperature and humidity combinations.

Table 1. Preferred drying temperatures based on RH% at 22.+ -. C.

Drying temperature (. Degree. C.)	Relative humidity (% -22 ℃ C.)
		110	>60
80-110	40-60
		60-80	15-40
10-50	<15％

Based on these findings, a new method for drying Solid Phase Microextraction (SPME) coatings, in particular biocompatible SPME coatings yielding BioSPME devices with consistent extraction efficiency and biocompatibility, is provided. The method includes determining a relative humidity (RH percentage or RH%) of the drying system measured at 22+ -2deg.C, selecting an appropriate drying temperature range based on the relative humidity, and introducing the coated device into the drying system; and maintaining the drying temperature in the drying system within the selected temperature range for a time sufficient to dry the coating. In certain embodiments, the drying system is a flow-through drying system. Such a drying system utilizes a drying gas of a flow-through system, which may be air, nitrogen or other inert gas.

According to the methods provided herein, the temperature of the drying system is selected based on the RH% of the drying gas at 22±2 ℃, i.e., for a flow-through drying system, the temperature of the drying gas. In a first embodiment, wherein RH% is greater than 60%, the drying temperature is maintained in the range of 110 ℃ to 160 ℃. In a second embodiment, wherein RH% is in the range of 40% to 60%, the drying temperature is maintained in the range of 80 ℃ to 110 ℃. In a third embodiment, wherein RH% is in the range of 15% to 40%, the drying temperature is maintained in the range of 60 ℃ to 80 ℃.

In a fourth embodiment, wherein RH% is less than 15%, the drying temperature is maintained in the range of 10 ℃ to 50 ℃.

The biocompatible coatings provided herein include a binder selected from the group consisting of Polyacrylonitrile (PAN), polyacrylamide, polyethylene glycol (PEG), polypyrrole, derivatized cellulose, polysulfone, polydimethylsiloxane (PDMS), polyacrylate, polytetrafluoroethylene (PTFE), and polyaniline, and an adsorbent selected from the group consisting of functionalized silica, carbon, polymer resins, and combinations thereof. In some embodiments, the adsorbent is a C18, C8 or mixed mode functionalized silica. In other embodiments, the adsorbent is a resin selected from the group consisting of HLB resins, divinylbenzene resins, styrene resins, and styrene-divinylbenzene copolymer resins. Various other features are described below.

SPME coatings useful in the methods provided herein include adhesives and adsorbents. In some applications, the binder and the adsorbent are biocompatible. Biocompatible means that the coating is compatible with the biological sample of interest and should not negatively interfere with the adsorptive properties of the SPME coating or otherwise cause interference in sampling or analysis.

Some non-limiting examples of binders that can be used for SPME include Polyacrylonitrile (PAN), polyethylene glycol (PEG), polypyrrole, derivatized cellulose, polysulfone, polyacrylamide, polyamide, polydimethylsiloxane (PDMS), polyacrylate, polytetrafluoroethylene (PTFE), and polyaniline. For some applications, the adhesive should also be biocompatible. Particularly suitable biocompatible binders include Polyacrylonitrile (PAN), polyethylene glycol (PEG), polypyrrole, derivatized cellulose, polysulfone, polyacrylamide, and polyamide. In a preferred embodiment, the adhesive is a biocompatible adhesive. In a particularly preferred embodiment, the biocompatible adhesive is PAN.

Adsorbents useful in the SPME devices described herein include microspheres, such as functionalized silica spheres, functionalized carbon spheres, polymer resins, mixed mode resins, and combinations thereof. In general, microspheres useful for liquid chromatography (i.e., affinity chromatography) and microspheres useful for Solid Phase Extraction (SPE) and Solid Phase Microextraction (SPME) are preferred for the coatings described herein.

In particular, the adsorbent may comprise functionalized silica microspheres, such as C18 silica (silica particles derivatized with a hydrophobic phase containing octadecyl), C8 silica (silica particles having a bonding phase containing octyl groups), RP-amide-silica (silica having a bonding phase containing palmitoylaminopropyl groups) or HS-F5-silica (silica having a bonding phase containing pentafluorophenyl-propyl groups).

Some other non-limiting examples of suitable adsorbents include: normal phase silica, C1 silica, C4 silica, C6 silica, C8 silica, C18 silica, C30 silica, phenyl/silica, cyano/silica, diol/silica, ionic liquid/silica, titan ^TM Silica (Millipore Sigma), molecularly imprinted polymer microparticles, hydrophilic-lipophilic-balance (HLB) microparticles (particularly those disclosed in co-pending U.S. patent application Ser. No. 16/640,575 published as US 2020/0197907),1006 (millipore sigma), poly (divinylbenzene), polystyrene, and poly (styrene-co-divinylbenzene). Mixtures of adsorbents can also be used in the coating. The adsorbents used in the coatings described herein can be inorganic (e.g., silica), organic (e.g., silica)Or divinylbenzene) or inorganic/organic mixtures (e.g., silica and organic polymers). In a preferred embodiment, the adsorbent is C18 silica, C8 silica or mixed mode functionalized silica. In a particularly preferred embodiment, the adsorbent is C18 silica.

The diameter of the adsorbent particles or microspheres may be in the range of about 10nm to about 1mm. In some embodiments, the spherical particles have a diameter in the range of about 20nm to about 125 μm. In certain embodiments, the diameter of the microspheres is in the range of about 30nm to about 85 μm. In some embodiments, the spherical particles have a diameter in the range of about 10nm to about 10 μm. Preferably, the spherical particles have a narrow particle size distribution.

In some embodiments, the surface area of the sorbent particles is about 10m ² /g to 1000m ² In the range of/g. In some embodiments, the porous spherical particles have a surface area of about 350m ² /g to about 675m ² In the range of/g. In some embodiments, the surface area is about 350m ² /g; in other embodiments, the surface area is about 375m ² In other embodiments, the surface area is about 400m ² /g; in other embodiments, the surface area is about 425m ² /g; in other embodiments, the surface area is about 450m ² /g; in other embodiments, the surface area is about 475m ² /g; in other embodiments, the surface area is about 500m ² /g; in other embodiments, the surface area is about 525m ² /g; in other embodiments, the surface area is about 550m ² /g; in other embodiments, the surface area is about 575m ² /g; in other embodiments, the surface area is about 600m ² /g; in other embodiments, the surface area is about 625m ² /g; in other embodiments, the surface area is about 650m ² /g; in still other embodiments, the surface area is about 675m ² /g; and in still other embodiments, a surface area of about 700m ² /g。

Preferably, the sorbent particles used in the devices described herein are porous. In some embodiments, the spherical particles have an average pore size of aboutTo about->Within a range of (2). In some embodiments, the porosity is about +.>To about->Within the scope of (1), in other embodiments, the porosity is about +.>To about->Within a range of (2). Furthermore, the average pore size of the spherical particles used herein may be about +.>About->About->About->About->AboutAbout->About->About->About->About->About->About->About->AboutAbout->About->About->About->About->About->Or about->

Coating layer

To apply the SPME coating on a substrate, a slurry comprising an adsorbent and a binder is prepared.

When the particles are silica particles and the biocompatible coating is PAN, the PAN to silica ratio may be between 1:0.5 and 1:7 (w/w). The preferred ratio of PAN/silica is 1:2 to 1:6 (w/w). The ratio is based on the net weight of silica and is adjusted to the phase loading on the silica particles. The PAN: solvent solution may be between about 5% (1:20) to about 15% (1:6.7) PAN (w/w). Preferably, the PAN: solvent solution may be between about 6% (1:16.7) to 12% (1:8.3) PAN (w/w). More preferably, the PAN: solvent solution may be about 7.5% (1:13.3) PAN (w/w). The solvent may be selected from Dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylamine (DMA), chloroacetonitrile, dioxanone, dimethyl phosphite, dimethylsulfone, gamma-butyrolactone, ethylene carbonate, nitric acid, sulfuric acid, and mixtures thereof. Preferably, the solvent is DMF.

In preparation for coating, a slurry of the adsorbent in the binder is prepared. The adsorbent, binder and solvent are weighed into a vessel. If desired, larger pieces or agglomerates of the adsorbent are crushed, for example with a doctor blade or mixer. The binder is dissolved in a solvent. Sonication and mixing may also be used to ensure a homogeneous distribution of particles in the binder solution. If desired, the slurry may be degassed prior to coating the substrate.

In the dip coating process, the substrate is lowered into the SPME coating slurry, then removed and dried according to the methods provided herein. Alternatively, a spraying method may be used in which the slurry is uniformly sprayed onto the substrate. For suitable substrates (e.g., fibers), a continuous coating process may be used.

According to the methods provided herein, the coating is dried in a temperature controlled environment, wherein the drying temperature is selected based on the humidity of the drying environment. In one embodiment, a flow-through drying system is used. The relative humidity (RH percentage or RH%) of the dry gas is measured at 22±2 ℃ or relative to that temperature. When drying the coating, a drying temperature selected based on% RH is maintained in the drying system. Suitable drying gases include air, nitrogen or other inert gases.

In a first embodiment, wherein RH% is greater than 60%, the drying temperature is maintained in the range of 110 ℃ to 160 ℃. In a second embodiment, wherein RH% is in the range of 40% to 60%, the drying temperature is maintained in the range of 80 ℃ to 110 ℃. In a third embodiment, wherein RH% is in the range of 15% to 40%, the drying temperature is maintained in the range of 60 ℃ to 80 ℃. In a fourth embodiment, wherein RH% is less than 15%, the drying temperature is maintained in the range of 10 ℃ to 50 ℃.

Instead, the humidity of the drying system may be selected based on the desired drying temperature. However, in most cases, the drying temperature is easier to control than the humidity level.

The coating thickness of the SPME coating may be varied to achieve the desired properties. In various embodiments, the coating thickness may be in the range of about 0.1 μm to about 200 μm. In a preferred embodiment, the coating thickness is in the range of about 2 μm to about 50 μm. In other embodiments, the coating thickness may be, for example, about 1 μm, about 2 μm, about 3 μm, about 4 μm, about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 55 μm, about 60 μm, about 65 μm, about 70 μm, about 75 μm, about 80 μm, about 90 μm, about 100 μm, about 110 μm, about 120 μm, about 130 μm, about 140 μm, about 150 μm, about 160 μm, about 170 μm, about 180 μm, about 190 μm, or about 200 μm. In some embodiments, the coating thickness is in the range of about 2 μm to about 50 μm, in other embodiments, the coating thickness is in the range of about 2 μm to about 40 μm, in still other embodiments, the coating thickness is in the range of about 5 μm to about 30 μm, in still other embodiments, the coating thickness is in the range of about 10 μm to about 100 μm. In a preferred embodiment, the coating thickness is in the range of about 10 μm to about 50 μm. For example, by performing the coating step multiple times, the coating thickness can be varied. For example, thinner coatings may be used when the sample size is very small or when rapid equilibrium extraction is required, however, thinner coatings may limit the amount of analyte that can be extracted. For a multi-pin arrangement, it is preferred that the coating thickness be uniform over all pins.

In some embodiments, the SPME coating is applied directly to the substrate without pretreatment. In other embodiments, the substrate may be pretreated prior to application of the SPME coating. When the substrate is plastic, the plastic substrate may be pretreated to roughen the surface to improve the adhesion of the SPME coating to the surface. Some conventional methods of roughening plastic surfaces include, for example, mechanical methods such as sand blasting, barreling, and grinding with a power tool; physical methods such as flame, corona discharge, plasma; or chemical methods such as acid etching, anodizing to enhance adhesion of the SPME coating to the substrate. In a preferred embodiment, the plastic substrate may be coated with a pre-coat to enhance the adhesion of the SPME coating to the substrate. Preferred precoats may include X18 (Master Bond, inc.) optionally including particles, such as silica, carbon or polymeric resins or PAN. When a precoat is used, the substrate is coated with the precoat, allowed to dry, then coated with the SPME coating, and then immersed in water for a time sufficient to form an SPME coated film. Such a precoat is described in more detail in the co-pending International application entitled "Pre-Coatings for BioSPME Devices" filed by the applicant Sigma-Aldrich Co.LLC at 12, 2021.

The methods described herein can be used to dry coatings on any device useful in SPME, including, for example, fibers, blades, tubes, screens or nets, columns, and pins. As used herein, the term "pin" includes a thin sheet of metal or plastic having a tip at one end. Such pins may be cylindrical, rod-shaped, conical, frustoconical, pyramidal, truncated pyramidal, rectangular, square, etc. The pins described herein preferably have a solid closed surface. When a pin is referred to as a "solid pin" or "wherein the pin is solid," it means that the surface of the pin is solid. A solid pin as defined herein may be distinguished from a design having an opening in the tip, such as may be used as a housing to hold SPE or SPME fibers, where typical metal fibers are substrates coated with SPE or SPME coatings. The surface of the pin was coated with an SPME coating. Since only the coated outer surface of the pin is in contact with the sample, it is not important that the inner surface is solid or hollow, as neither the coating nor the sample is in contact with the inner surface. The tip or tip (point) of the pin may be flat, rounded or may be a point. In some embodiments, the SPME device may include a single pin, while in other embodiments, the device may include multiple pins. Particularly preferred pin arrangements are described in co-pending international publication number WO 2019/036414, the entire contents of which are incorporated herein by reference.

Preferably, the diameter of the pin is in the range of about 0.2mm to about 5mm. In a preferred embodiment, the diameter of the pin is in the range of about 0.5mm to about 2 mm. In a particularly preferred embodiment, the diameter of the pin is about 1mm. The length of the pin may be varied, for example, to accommodate various sample volumes and hole depths. The length of the pin is preferably in the range of about 0.2mm to about 5cm. In some embodiments, the length may be about 0.5mm to about 2.5cm. In other embodiments, the length may be about 1mm to about 1cm.

The pins may be made of any suitable material including, for example, plastic, metal, glass, ceramic, and the like. In a preferred embodiment, the pin is made of plastic. Some non-limiting examples of suitable plastics for SPME substrates (e.g., pins) include, but are not limited to, polyolefin, polyamide, polycarbonate, polyester, polyurethane, polyvinylchloride, polytetrafluoroethylene (PTFE), polyetheretherketone (PEEK), polysulfone, and polybutylene terephthalate substrates. In some preferred embodiments, the plastic pin is polypropylene or polyethylene.

The coatings, precoats, and SPME coatings described herein are applied to the ends of pins that will contact the sample of interest. In some embodiments, the pin is coated with a precoat and SPME coating for about half the length. In other embodiments, the pin is coated with a precoat and SPME coating for about one-quarter of its length. In various embodiments, the precoat and SPME coating may cover some portion of the length of one or more pins, for example, 1/10, 1/5, 1/4, 1/3, or 1/2 of the length of one or more pins. In other embodiments, the coating may be measured from the tip of the pin (i.e., the end of the pin that will contact the sample). In some embodiments, the pre-coat and coating may cover 1mm pins, in other embodiments, the pre-coat and coating may cover 1.5mm pins, and in other embodiments, the pre-coat and coating may cover 2mm pins. In an embodiment of a 1cm pin, the pre-coat and coating may cover 0.5mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm, 1mm, 1.1mm, 1.2mm, 1.3mm, 1.4mm, 1.5mm, 1.6mm, 1.7mm, 1.8mm, 1.9mm, 2mm, 2.1mm, 2.2mm, 2.3mm, 2.4mm, 2.5, 2.6mm, 2.7mm, 2.8mm, 2.9mm, 3mm, 3.1mm, 3.2mm, 3.3mm, 3.4mm, 3.5mm, 3.6mm, 3.7mm, 3.8mm, 3.9mm, 4.1mm, 4.2mm, 4.3mm, 4.4mm, 4.5mm, 4.6mm, 4.7mm, 4.8mm or 5mm from the end of the pin. In other embodiments, other suitable coating coverage may be readily determined based on the length, shape, and diameter of the pin.

When the device comprises more than one pin, for example 4 pins, 8 pins, 12 pins, 16 pins, 24 pins, 48 pins, 96 pins, 384 pins or 1536 pins, it is preferred that the coating covers similar parts of each pin. In one embodiment, the pins of the multi-pin device are coated simultaneously using a dip coating process. In such a method, the plastic multi-pin device is first immersed in the precoat, removed and allowed to dry, then immersed in the SPME coating, removed and dried using the methods provided herein. Only the portion of the pin to be coated is contacted with the coating formulation or slurry. Such a coating method may ensure consistent coating on all pins in the device. Alternatively, other coating methods may be used, such as spray coating or continuous coating. In both single and multi-pin embodiments, dip coating is the most preferred method of applying the pre-coat and SPME coating to the plastic substrate/pin.

SPME coatings prepared using the methods described herein were visually inspected, tested for asperities (ruggeddess) and adhesion, and evaluated for extraction efficiency and protein binding. Exemplary methods of these evaluations are summarized below.

The dried coating was visually observed using an optical microscope and/or SEM. The coatings were tested for asperity and adhesion as follows: (a) By finger rubbing over the cured coating, and (b) by blue tape adhesion test. The blue tape adhesion test was performed as follows: a blue paint tape (medium tack) was applied to the coated cured SPME device and allowed to stand in place for 90 seconds, then the tape was removed at an angle of 180 degrees relative to the device. Adhesion was observed visually using a microscope.

To test extraction efficiency, 96 pin SPME devices were coated with a PAN/C18 SPME coating and dried using the methods described herein. SPME devices were tested using the following extraction procedure.

Conditioning: in Nunc 1mL 96-well plates, 800. Mu.L isopropanol was run at about 1200rpm for 20 minutes.

Washing: in Nunc 1mL 96-well plates, 800. Mu.L water was run at about 1200rpm for 10 seconds.

Extraction: on a shaker, in Nunc 1mL 96-well plates, at about 1200rpm, in 800. Mu.L buffer for 30 minutes. The admixture (spike) was prepared with carbamazepine in PBS buffer (ph=7.48) at 5000 ng/mL. The organic content percentage is 0.5%. Content at room temperature.

Washing: in Nunc 1mL 96-well plates, 800. Mu.L water was run at about 1200rpm for 10 seconds.

And (3) desorption: 600. Mu.L of a conical 96-well plate was used with Axygen at about 1200rpm in 400. Mu.L 80:20 methanol:water for 20 minutes.

A robot system: apricot

The pin tool was analyzed on HPLC with UV detection using the parameters in table 2.

Table 2. HPLC parameters for measuring extraction efficiency.

Protein binding extraction procedure. Protein binding was tested using the following extraction procedure.

Conditioning: in Nunc 1mL 96-well plates, static, in 800. Mu.L isopropanol for 20 min.

Washing: in Nunc 1mL 96-well plates, static, in 800. Mu.L water for 10 seconds.

Extraction: with the adapter, in Nunc 1mL conical 96 well plate, at about 1200rpm, in 800. Mu.L 100ng/mL buffer or plasma/serum for 30 minutes. The temperature was set to 37 ℃.

Washing: in Nunc 1mL 96-well plates, static, in 800. Mu.L water for 60 seconds.

And (3) desorption: 600. Mu.L of the conical 96-well plate was used with Axygen, and the plate was static in 400. Mu.L of 80:20 methanol in water for 20 minutes.

A robot system: hamilton

Protein binding LC/MS methods were performed on an Agilent 1290/AB Sciex 650Q Trap using the conditions in Table 3.

Table 3 LC/MS conditions for protein binding assays.

Using the analytical methods described above, it was found that coatings prepared using the methods described herein have good extraction efficiencies and are more consistent than coatings prepared using conventional methods. While the above-described evaluation methods are performed using 96 pin devices, these exemplary methods are not limited to such devices, but may also be used with other SPME devices.

Examples

Example 1. Preparation of BioSPME coating slurry and coating of SPME devices in PAN for C18. 40.0g of PAN was weighed into 500.0g of DMF. The PAN was broken into small pieces using a doctor blade. The mixture was incubated at 85℃until dissolved.

132g of C18 was weighed into a PAN/DMF solution. The mixture was thoroughly mixed with a spatula, and the resulting slurry was then roll-mixed for 60 minutes. The slurry was then sonicated (mixture) for 20 minutes and then homogenized for 45 minutes. The process is then repeated. The resulting slurry was degassed and then mixed until ready for coating.

Example 2. SPME slurry coating apparatus of example 1 was used and dried at a constant temperature of 110 ℃ between 20% Relative Humidity (RH), 39% RH, about 48% RH, and about 60-70% RH. The extraction efficiency is shown in table 4.

Table 4. Extraction efficiencies of coatings dried at 110℃at different RH%.

Sample of	01082020-2	02042020-22	02042020-39	08302019
					RH％	20	39	About 48	About 60 to about 70
Extraction efficiency	About 0.3	About 0.6 to 0.7	About 0.9	About 1.1

Fig. 1 shows SEM images of PAN/C18 coatings dried at 110 ℃ at different humidity levels. When the relative humidity was changed from 20% to 70%, the extraction efficiency of the coating was changed from 0.3 to 1.1 as shown in table 4.

Example 3 to further investigate the effect of humidity levels on drying, SPME devices were coated with the coating of example 1. The drying temperature was kept constant at 22 ℃ and the relative humidity percentage varied between 39% and 7%. Humidity levels, extraction efficiencies, and protein binding are shown in table 5, and SEM images are shown in fig. 2.

Table 5. Extraction efficiency and protein binding of PAN/C18 coatings dried at 22℃at different humidity levels.

When the PAN/C18 coating was dried at 22 ℃ at different humidity levels, the morphology of the coating varied significantly, as shown in fig. 2. In addition, the extraction efficiency of the coating decreases with decreasing relative humidity. Furthermore, the coatings dried only at low humidity levels (at 22 ℃ below 15 RH%) show good biocompatibility. When the coating is dried at 22 ℃ at RH% of greater than 15%, the coating is susceptible to contamination by the plasma matrix and produces inaccurate protein binding values. Protein binding was significantly higher for device 02042020-3RT dried at 22 ℃ and 39% RH than for the reference protein (70-80%). Whereas the protein binding of device 02202020-2RT with the coating prepared by the method described herein, dried at 22 ℃ and 10% RH, was very consistent with the reference protein binding.

The PAN/C18 coating dried at high temperature (e.g. 110 ℃) shows good biocompatibility. However, in order to ensure the efficacy of the coating, the humidity of the drying step must be high (> 60%). PAN/C18 coatings dried at low temperatures (e.g. 22 ℃) show good efficacy. However, in order to ensure biocompatibility of the coating, the humidity of the drying step must be low (< 15%). The biocompatibility and efficacy of PAN/C18 coatings vary with both drying temperature and humidity. In order to ensure biocompatibility and efficacy of the PAN/C18 coating, the drying temperature and humidity must be controlled. Preferred drying temperatures based on RH% are listed in Table 1.

Example 4. PAN/C18 slurry was prepared as in example 1. The 96-pin device was Pre-treated as disclosed in the co-pending international application entitled "Pre-Coatings for BioSPME Devices" filed by the applicant Sigma-Aldrich Co.LLC at 12.2 of 2021. A pretreatment apparatus dip-coated with PAN/C18 slurry. The dip-coating conditions were: and (3) the following steps: 0.25mm/s, the following: 1mm/s, residence time: 3 seconds, dipping: 4.95mm rake rack (rake rest): 15 seconds.

Example 5. Two 96 pin devices were coated with the slurry of example 4. One was dried at 60 ℃,34% RH; the second was dried at 80℃and 35% RH. Protein binding of carbamazepine was measured using the method described above. For conventional SPME devices, carbamazepine reference protein binding is 70-80%. The results are summarized in table 6 below.

Table 6.2 protein binding by pin tool.

Protein binding of two devices with coatings prepared by the methods described herein was very consistent with reference protein binding.

Claims (11)

1. A method for drying a coating suitable for biocompatible solid phase microextraction (BioSPME) in a flow-through drying system, the method comprising:

determining the relative humidity (RH%) of the drying system at 22+ -2deg.C,

a suitable drying temperature range is selected for the drying system based on the RH% of the drying system,

adjusting the temperature of the drying system within a selected temperature range;

introducing a device comprising a BioSPME coating into the drying system; and

the temperature of the drying system is maintained within the selected temperature range for a time sufficient to dry the coating.

2. The method of claim 1, wherein the drying temperature is maintained in the range of 110 ℃ to 160 ℃ when rh% is greater than 60%.

3. The method of claim 1, wherein the drying temperature is maintained in the range of 80 ℃ to 110 ℃ when RH is in the range of 40% to 60%.

4. The method of claim 1, wherein the drying temperature is maintained in the range of 60 ℃ to 80 ℃ when RH is in the range of 15% to 40%.

5. The method of claim 1, wherein the drying temperature is maintained in the range of 10 ℃ to 50 ℃ when RH is less than 15%.

6. The method of any one of claims 1-5, wherein the drying system comprises a drying gas, and the drying gas is selected from the group consisting of air, nitrogen, and other inert gases.

7. The method of any one of claims 1-6, wherein the biocompatible coating comprises a binder and an adsorbent.

8. The method of claim 7, wherein

The binder is selected from the group consisting of Polyacrylonitrile (PAN), polyacrylamide, polyethylene glycol (PEG), polypyrrole, derivatized cellulose and polysulfone, polydimethylsiloxane, polyacrylate, polytetrafluoroethylene and polyaniline; and

the adsorbent is selected from the group consisting of functionalized silica, carbon, polymeric resins, and combinations thereof.

9. The method of any one of claims 7 or 8, wherein the binder comprises PAN and the adsorbent comprises C18, C8, or mixed mode functionalized silica.

10. The method of any one of claims 7 or 8, wherein the adsorbent is a polymer resin selected from the group consisting of an HLB resin, a divinylbenzene resin, a styrene resin, a divinylbenzene-co-styrene resin, and combinations thereof.

11. A device for solid phase microextraction, prepared by the method of any one of claims 1-10.