Demonstration of direct bioanalysis of drugs in plasma using nanoelectrospray infusion from a silicon chip coupled with tandem mass spectrometry

Quantitative bioanalysis by direct nanoelectrospray infusion coupled to tandem mass spectrometry has been achieved using an automated liquid sampler integrated with an array of microfabricated electrospray nozzles allowing rapid, serial sample introduction (1 min/ sample). Standard curves prepared in human plasma for verapamil (r2 = 0.999) and its metabolite norverapamil (r2 = 0.998) were linear over a range of 2.5-500 ng/ mL. Based on the observed precision and accuracy, a lower limit of quantitation of 5 ng/mL was assigned for both analytes. Sample preparation consisted of protein precipitation with an organic solvent containing the structural analogue gallopamil as an internal standard. Protein precipitation was selected both to maximize throughput and to test the robustness of direct nanoelectrospray infusion. Aliquots of supernatant (10 pL) were transferred to the back plane of the chip using disposable, conductive pipet tips for direct infusion at a flow rate of 300 nL/min. Electrospray ionization occurred from the etched nozzles (30-microm o.d.) on the front of the chip, initiated by a voltage applied to the liquid through the pipet tip. The chip was positioned near the API sampling orifice of a triple quadrupole mass spectrometer, which was operated in selected reaction monitoring mode. Results are presented that document the complete elimination of system carry-over, attributed to lack of a redundant fluid path. This technology offers potential advantages for MS-based screening applications in drug discovery by reducing the time for methods development and sample analysis.     

Electroosmosis-based nanopipettor

Decreasing the volume of reagent solutions consumed in each assay is an effective means to reduce the overall cost in high-throughput analysis laboratories. Recently, increasing attention has been paid to investigate the behavior of individual cells. If one wishes to transfer solution to or from a single cell, a picoliter pipettor is needed since the entire cell volume is commonly less than 1 nL. While pressure ejection and iontophoresis have been used to deliver picoliter volumes of solutions, these techniques cannot yield routine pipettors which perform both solution "picking up" and "dispensing" functions. The state-of-the-art pipettors can handle liquids down to approximately 100 nL, although the pipetting accuracy and precision deteriorate considerably from microliters to nanoliters. If one wishes to pipet reagents of less than 100 nL, new pipettors need to be developed. Electroosmosis has been utilized to pump solutions at flow rates of nanoliters to approximately picoliters per second, which is ideal for nanopipettors. The issue is how to arrange fluidic/electrical connections so that pipetting functions can be performed conveniently. In this paper, we present the results of our initial attempt to develop an electroosmosis-based nanopipettor. The first version of this pipettor consists of a microfabricated electroosmotic (EO) flow pump, a polyacrylamide grounding interface, and a nanoliter-to-picoliter pipet tip. The detailed configuration and fabrication process of the pipettor are discussed. An excellent feature of an EO-driven pipettor is that it has no moving parts. Good reproducibilities (RSD = 6% at 140 pL, 2% at 950 pL, and 2% at 13 nL) and accuracies (9% at 0.13 nL, 4% at 1.0 nL, and 3% at 10 nL) of this pipettor have been demonstrated to aliquot/transport nanoliter-to-picoliter solutions.     

Stop and go extraction tips for matrix-assisted laser desorption/ionization,nanoelectrospray, and LC/MS sample pretreatment in proteomics

Proteomics is critically dependent on optimal sample preparation. Particularly, the interface between protein digestion and mass spectrometric analysis has a large influence on the overall quality and sensitivity of the analysis. We here describe a novel procedure in which a very small disk of beads embedded in a Teflon meshwork is placed as a microcolumn into pipet tips. Termed Stage, for STop And Go Extraction, the procedure has been implemented with commercially available material (C18 Empore Disks (3M, Minneapolis, MN)) as frit and separation material. The disk is introduced in a simple and fast process yielding a convenient and completely reliable procedure for the production of self-packed microcolumns in pipet tips. It is held in place free of obstacles solely by the narrowing tip, ensuring optimized loading and elution of analytes. Five disks are conveniently placed in 1 min, adding <0.1 cent in material costs to the price of each tip. The system allows fast loading with low backpressure (>300 micro/min for the packed column using manual force) while eliminating the possibility of blocking. The loading capacity of C18-StageTips (column bed: 0.4 mm diameter, 0.5 mm length) is 2-4 microg of protein digest, which can be increased by using larger diameter or stacked disks. Five femtomole of tryptic BSA digest could be recovered quantitatively. We have found that the Stage system is well-suited as a universal sample preparation system for proteomics.     

Openly accessible microfluidic liquid handlers for automated high-throughput nanoliter cell culture

Cell culture is typically performed in Petri dishes, with a few million cells growing together, or in microwell plates with thousands of cells in each compartment. When the throughput of each experiment, especially of screening based assays, is increased, even using microliter solution per well will cost a considerable amount of cells and reagents. We took a rational approach to reduce the volume of each cell culture chamber. We designed and fabricated a poly(dimethylsiloxane) based liquid pipet chip to deliver and transfer nanoliter (50-500 nL) samples and reagents with high accuracy and robustness. A few tens to a few hundreds of cells can be successfully seeded, transferred, passaged, transfected, and stimulated by drugs on a microwell chip using this pipet chip automatically. We have used this system to test the cell growth dynamically, observed the correlation between the culture conditions and cell viabilities, and quantitatively evaluated cell apoptosis induced by cis-diammineplatinum(II) dichloride (cisplatin). This system shows great potential to facilitate large-scale screening and high-throughput cell-array based bioassays with the volume of each individual cell colony at the nanoliter level.     

Voltammetry at micropipet electrodes

The use of micropipet electrodes for quantitative voltammetric measurements of ion-transfer (IT) and electron-transfer (ET) reactions at the interface between two immiscible electrolyte solutions (ITIES) requires knowledge of geometry of the liquid interface. The shape of the meniscus formed at the pipet tip was studied in situ by video microscopy under controlled pressure. The shape of the interface can be changed from a complete sphere to a concave spherical cap by varying the pressure applied to the pipet, and the diffusion current to the pipet changes accordingly. With no external pressure applied, the water/organic interface turned out to be flat, and the voltammetric response of a pipet must follow the well-known theory for a microdisk electrode. The large deviations from this theory observed previously can be attributed to a small amount of the filling aqueous solution which escapes from the pipet and forms a thin layer on its outer wall. This effect can be eliminated by making the outer pipet wall hydrophobic. Procedures have been developed for independent silanization of the inner and outer walls of the pipet. Pipets with a silanized inner wall can be filled with an organic solvent (e.g., 1,2-dichloroethane) and be used for voltammetric measurements in aqueous solutions. Another mode of voltammetry is based on trapping of a thin layer of organic solvent in the narrow shaft of a pipet between the filling solution and the aqueous outer phase. This arrangement is potentially useful for electrochemical catalysis and sensor applications.     

Determination of beta-2 microglobulin levels in plasma using a high-throughput mass spectrometric immunoassay system

A high-throughput mass spectrometric immunoassay (MSIA) system for the analysis of proteins directly from biological fluids is reported. A 96-well-format robotic workstation equipped with antibody-derivatized affinity pipet tips was used for the parallel extraction of specific proteins from samples and subsequent deposition onto 96-well arrayed matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) targets. Interferences from nonspecifically bound proteins were minimized through choice of appropriate affinity pipet tip derivatization chemistries. Sample preparation for MALDI-TOFMS was enhanced through the use of hydrophobic/hydrophilic contrasting targets, which also presented functionalities found to promote matrix/analyte crystal growth. Automated mass spectrometry was used in the unattended acquisition of data, resulting in an analysis rate of approximately 100 samples/h (biological fluid-->data). The quantitative MSIA of beta2m levels present in human plasma samples is given as illustration.     

Specificity of immobilized metal affinity-based IMAC/C18 tip enrichment of phosphopeptides for protein phosphorylation analysis

We have developed a simple, highly specific enrichment procedure for phosphopeptides, by increasing the specificity of an immobilized metal affinity column (IMAC) without using any chemical reaction. The method employs a biphasic IMAC-C18 tip, in which IMAC beads are packed on an Empore C18 disk in a 200-microL pipet tip. Phosphopeptides are separated from non-phosphopeptides on the IMAC in an optimized solvent without any chemical reaction, then desorbed from the IMAC using a phosphate buffer, reconcentrated, and desalted on the C18 disk. The increase in selectivity was achieved by (a) using a strong acid to discriminate phosphates from carboxyl groups of peptides and (b) using a high concentration of acetonitrile to remove hydrophobic non-phosphopeptides. The entire procedure was optimized by using known phosphoproteins such as Akt1 kinase and protein kinase A. Although it was difficult to detect phosphopeptides in MALDI-MS spectra of tryptic peptide mixtures before enrichment, after the IMAC procedure, we could successfully detect phosphopeptides with almost no non-phosphopeptides. Next, we constructed an array of IMAC-IMAC/C18 tips, such that number of arrayed tips on a 96-well plate could easily be changed depending on the loading amount of sample. Applying this approach to mouse forebrain resulted in the identification of 162 phosphopeptides (166 phosphorylation sites) from 135 proteins using nano-LC/MS.     

Sample preparation for the analysis of complex carbohydrates by multicapillary gel electrophoresis with light-emitting diode induced fluorescence detection

This paper evaluates various sample preparation methods for multicapillary gel electrophoresis based glycan analysis to support electrokinetic injection. First the removal of excess derivatization reagent is discussed. Although the Sephadex G10 filled multiscreen 96-well filter plate and Sephadex G10 filled pipet tips enabled increased analysis sensitivity, polyamide DPA-6S pipet tips worked particularly well. In this latter case an automated liquid handling system was used to increase purification throughput, necessary to feed the multicapillary electrophoresis unit. Problems associated with the high glucose content of such biological samples as normal human plasma were solved by applying ultrafiltration. Finally, a volatile buffer system was developed for exoglycosidase-based carbohydrate analysis.     

Simple reconstitution of protein pores in nano lipid bilayers

We developed a new, simple and robust approach for rapid screening of single molecule interactions with protein channels. Our glass nanopipets can be fabricated simply by drawing glass capillaries in a standard pipet puller, in a matter of minutes, and do not require further modification before use. Giant unilamellar vesicles break when in contact with the tip of the glass pipet and form a supported bilayer with typical seal resistances of ～140 GΩ, which is stable for hours and at applied potentials up to 900 mV. Bilayers can be formed, broken, and re-formed more than 50 times using the same pipet enabling rapid screening of bilayers for single protein channels. The stability of the lipid bilayer is significantly superior to that of traditionally built bilayers supported by Teflon membranes, particularly against perturbation by electrical and mechanical forces. We demonstrate the functional reconstitution of the E. coli porin OmpF and α-hemolysin in a glass nanopipet supported bilayer. Interactions of the antibiotic enrofloxacin with the OmpF channel have been studied at the single-molecule level, demonstrating the ability of this method to detect single molecule interactions with protein channels. High-resolution conductance measurements of protein channels can be performed with low sample and buffer consumption. Glass nanopipet supported bilayers are uniquely suited for single-molecule studies as they are more rigid and the lifetime of a stable membrane is on the scale of hours, closer to that of natural cell membranes.     

Microfluidic chip for peptide analysis with an integrated HPLC column, sample enrichment column, and nanoelectrospray tip

Current nano-LC/MS systems require the use of an enrichment column, a separation column, a nanospray tip, and the fittings needed to connect these parts together. In this paper, we present a microfabricated approach to nano-LC, which integrates these components on a single LC chip, eliminating the need for conventional LC connections. The chip was fabricated by laminating polyimide films with laser-ablated channels, ports, and frit structures. The enrichment and separation columns were packed using conventional reversed-phase chromatography particles. A face-seal rotary valve provided a means for switching between sample loading and separation configurations with minimum dead and delay volumes while allowing high-pressure operation. The LC chip and valve assembly were mounted within a custom electrospray source on an ion-trap mass spectrometer. The overall system performance was demonstrated through reversed-phase gradient separations of tryptic protein digests at flow rates between 100 and 400 nL/min. Microfluidic integration of the nano-LC components enabled separations with subfemtomole detection sensitivity, minimal carryover, and robust and stable electrospray throughout the LC solvent gradient.     

Selective extraction and characterization of a histidine-phosphorylated peptide using immobilized copper(II) ion affinity chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Phosphorylation is the predominant posttranslational modification involved in regulating enzymatic activity and mediating signal transduction in prokaryotic and eukaryotic cells. Selective enrichment of phosphorylated peptides prior to mass spectrometric analysis facilitates identification of phosphorylated proteins, determination of specific phosphorylated residues, and characterization of the conditions under which phosphorylation occurs. Such protocols have been established for peptides containing residues that form phosphoesters, such as serine and threonine, using immobilized metal-ion affinity chromatography. Despite the importance of histidine phosphorylation in two-component signal transduction pathways, similar protocols for peptides containing phosphorylated histidine (P-His) residues have proven elusive, due to the instability of these modifications and the propensity of unphosphorylated histidines to interact with immobilized metals ions. We describe a method for the selective extraction of a P-His-containing peptide using immobilized copper(II) ions and disposable metal-chelating pipet tips (ZipTipMC, Millipore). The method is contingent upon pH-dependent interactions between the phosphate group and immobilized copper(II) ions. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with postsource decay confirms the identity and phosphorylation state of the extracted peptide. Peptides containing unphosphorylated histidine residues or other phosphorylated amino acids are not retained, demonstrating the specificity of the method for P-His-containing peptides.     

High-efficiency nanoscale liquid chromatography coupled on-line with mass spectrometry using nanoelectrospray ionization for proteomics

We describe high-efficiency (peak capacities of approximately 10(3)) nanoscale (using column inner diameters down to 15 microm) liquid chromatography (nanoLC)/low flow rate electrospray (nanoESI) mass spectrometry (MS) for the sensitive analysis of complex global cellular protein enzymatic digests (i.e., proteomics). Using a liquid slurry packing method with carefully selected packing solvents, 87-cm-length capillaries having inner diameters of 14.9-74.5 microm were successfully packed with 3-microm C18-bonded porous (300-A pores) silica particles at a pressure of 18,000 psi. With a mobile-phase delivery pressure of 10,000 psi, these packed capillaries provided mobile-phase flow rates as low as approximately 20 nL/min at LC linear velocities of approximately 0.2 cm/s, which is near optimal for separation efficiency. To maintain chromatographic efficiency, unions with internal channel diameters as small as 10 microm were specially produced for connecting packed capillaries to replaceable nanoESI emitters having orifice diameters of 2-10 microm (depending on the packed capillary dimensions). Coupled on-line with a hybrid-quadrupole time-of-flight MS through the nanoESI interface, the nanoLC separations provided peak capacities of approximately 10(3) for proteome proteolytic polypeptide mixtures when a positive feedback switching valve was used for quantitatively introducing samples. Over a relatively large range of sample loadings (e.g., 5-100 ng, and 50-500 ng of cellular proteolytic peptides for 14.9- and 29.7-microm-i.d. packed capillaries, respectively), the nanoLC/nanoESI MS response for low-abundance components of the complex mixtures was found to increase linearly with sample loading. The nanoLC/nanoESI-MS sensitivity also increased linearly with decreasing flow rate (or approximately inversely proportional to the square of the capillary inner diameter) in the flow range of 20-400 nL/min. Thus, except at the lower loadings, decreasing the separation capillary inner diameter has an effect equivalent to increasing sample loading, which is important for sample-limited proteomic applications. No significant effects on recovery of eluting polypeptides were observed using porous C18 particles with surface pores of 300-A versus nonporous particles. Tandem MS analyses were also demonstrated using the high-efficiency nanoLC separations. Chromatographic elution time, MS response intensity, and mass measurement accuracy was examined between runs with a single column (with a single nanoESI emitter), between different columns (same and different inner diameters with different nanoESI emitters), and for different samples (various concentrations of cellular proteolytic peptides) and demonstrated robust and reproducible sensitive analyses for complex proteomic samples.     

Capillary electrophoresis-electrospray mass spectrometry for the characterization of high-mannose-type N-glycosylation and differential oxidation in glycoproteins by charge reversal and protease/glycosidase digestion.

The characterization of high-mannose-type N-glycosylation by capillary electrophoresis-electrospray mass spectrometry (CE-ESI MS) was described. In addition to the use of a cationic noncovalent capillary coating, strong acidic buffer, and charge reversal to increase the glycoform resolving power, N-glycosidase F (PNGase F) combined with a basic protease and alpha-mannosidase combined with an acidic protease were used to analyze the high-mannose-type N-glycosylation in ribonuclease B (RNase B) and in a novel C-type lectin from the venom of Trimeresurus stejnegeri (TSL). The structures of oligosaccharide, glycosylation sites, and glycoform distributions were determined simultaneously, and the differential oxidation of Met residues in glycopeptides obtained from TSL protease digestion was also characterized successfully by CE-MS/MS. The results showed that the oligosaccharide attached to RNase B has a structure of GlcNAc2Man5 approximately 9, and that attached to TSL has a structure of GlcNAc2Min5 approximately 8. The glycoform distributions in these glycoproteins are quite different, with the GlcNAc2Man5 type predominant in RNase B, and the GlcNAc2Man8 type, in TSL This method may be useful not only for the characterization of glycosylation sites and glycan structures, but also for the determination of the relative abundance of individual glycoforms.     

Attenuated total reflection-infrared nanofluidic chip with 71 nL detection volume for in situ spectroscopic analysis of chemical reaction intermediates

We present a micromachined silicon attenuated total reflection-infrared (ATR-IR) crystal with integrated nanofluidic glass channels which enables infrared spectroscopic studies with only 71 nL sample volume. Because of the short path length through silicon, the system allows IR spectroscopy down to 1200 cm(-1), which covers the typical fingerprint region of most organic compounds. To demonstrate proof-of-principle, the chip was used to study a Knoevenagel condensation reaction between malononitrile and p-anisaldehyde catalyzed by different concentrations of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in solvent acetonitrile. By in situ measurement, it was demonstrated for the first time that at certain concentrations of DBU, reaction intermediates become stabilized, an effect that slows down or even stops the reaction. This is thought to be caused by increased ionic character of the solvent, in which protonated DBU stabilizes the intermediates. This clearly demonstrates that infrared mechanistic studies of chemical reactions are feasible in volumes as little as 71 nL.     

Selective sampling of phosphopeptides for detection by MALDI mass spectrometry

The analysis of phosphopeptides by mass spectrometry (MS) is one of the most challenging tasks in proteomics. This is due to the lower isoelectric point (pI) of phosphopeptides, which leads to inefficient sample ionization in MS, particularly when competing with other peptides. The problem is compounded by the typical low abundance of phosphopeptides in biological samples. We describe here a simple nonsorptive method to isolate phosphopeptides based on their pI. A voltage is applied to selectively migrate the phosphopeptides into a capillary, which are negatively charged at acidic pH. The selectively sampled fraction is directly deposited onto MALDI sample target in nanoliter volumes (7-35 nL) for highly sensitive MS detection. No significant sample loss is evident in this procedure; hence, the MS was able to detect the isolated phosphopeptides at trace quantity. In this case, attomole-level detection limit is achieved for synthetic phosphopeptides (nM concentration and nL volume), from a mixture containing other peptides at up to 1 million times higher in concentration. Selective sampling was also applied to the tryptic digest of beta- and alpha-caseins to reveal the multiple phosphorylated peptides at the low-femtomole level using MALDI MS. Knowledge of pI based on the rejection/injection of peptides was found to be useful in peak assignment. To confirm the sequence of the selectively sampled peptides, fraction collection was performed for offline ESI MS/MS analysis.     

High-pressure microfluidic control in lab-on-a-chip devices using mobile polymer monoliths

We have developed a nonstick polymer formulation for creating moving parts inside of microfluidic channels and have applied the technique to create piston-based devices that overcome several microfluidic flow control challenges. The parts were created bycompletely filling the channels of a glass microfluidic chip with the monomer/ solvent/initiator components of a nonstick photopolymer and then selectively exposing the chip to UV light in order to define mobile pistons (or other quasi-two-dimensional shapes) inside the channels. Stops defined in the substrate prevent the part from flushing out of the device but also provide sealing surfaces so that valves and other flow control devices are possible. Sealing against pressures greater than 30 MPa (4,500 psi) and actuation times less than 33 ms are observed. An on-chip check valve, a diverter valve, and a 10-nL pipet are demonstrated. This valving technology, coupled with high-pressure electrokinetic pumps, should make it possible to create a completely integrated HPLC system on a chip.     

Programmable delivery of DNA through a nanopipet

We report the pulsed delivery of single-stranded DNA molecules through a nanopipet. The conical geometry of the pipet leads to a localized electric field, since all of the potential drop occurs in the tip region. Pulsatile delivery of DNA molecules can be achieved in an experimentally simple way with high precision by controlling the applied voltage. Single-molecule detection and fluorescence correlation spectroscopy in the nanopipet enable us to determine the number of molecules delivered. Anomalous slow diffusion of the DNA molecules in the pipet has also been observed. This nanopumping technique may have potential applications in local drug delivery and nanofabrication of biomolecules on surfaces in aqueous environments.     

Study of eye drops dispensing and dose variability by using plastic dropper tips

The application of eye drops from flexible dropper bottles fitted with different types of dropper tips is associated with the high variability of eye drop weights. The aim of this report was to investigate the simultaneous effect of three factors influencing the mean weight of drops dispensing from two plastic dropper tips. Using a designed experiment (Box-Behnken), the effect of the concentration of benzalkonium chloride solutions (BAC) in the range of 0-0.02%, the dispensing angle from 90° to 30° from horizontal, and the residual volume of liquid in the dropper bottle from 4 to 10 mL on the mean drop weights were examined. The significant effect of the increase in BAC concentration resulted in a linear decrease in drop weights for both of the dropper tips investigated. The significant effect of the dispensing angle was influenced by the dropper tip design. For the dropper tip A, the effect of the dropper tip tilt was described by the quadratic equation with a minimum, which corresponded to the dispensing angle equal to that of 48° from horizontal. Below this angle, the increase in drop weights occurred due to the drop formation from the wetted external surface of the tip orifice. The linear decrease in drop weights in response to the decrease in dispensing angle was detected for the dropper tip B. The regression equations and the contour line plots obtained allowed the drop weights to be estimated for the actual combinations of both the BAC concentration and the dispensing angle. The effect of the residual volume was found to be non-significant. Based on the formula of Tate's law, the direct proportion between surface tension of a solution and the radius of the effective perimeter of a dropper tip can be used to estimate the theoretical maximal weight of drops at the dispensing angle of 90°. Using the stalagmometric values of surface tension of the BAC solutions, the maximal drop weights were estimated for both of the dropper tips investigated. A comparison between the theoretical and the experimentally measured drop weights enabled the dropper tips behavior to be discussed by using Harkins and Brown correction factor F. The F-value of 0.74 noted for the dropper tip A differed from that of stalagmometer F-value (0.61) indicating a deviation from the simple drop formation process in answer to more complicated design of the dropper tip A. On the other hand, the F-value of 0.6 observed for the dropper tip B demonstrated the better consistency with stalagmometry. As a result, the dropper tip B with the linear decrease of drop weights in response to the increased concentration of BAC and the decreased dispensing angle without the adverse external drop formation could be recommended in real drop dispensing.     

Cotton HILIC SPE microtips for microscale purification and enrichment of glycans and glycopeptides

Solid-phase extraction microtips are important devices in modern bioanalytics, as they allow miniaturized sample preparation for mass spectrometric analysis. Here we introduce the use of cotton wool for the preparation of filter-free HILIC SPE microtips. To this end, pieces of cotton wool pads (approximately 500 μg) were packed into 10 μL pipet tips. The performance of the tips was evaluated for microscale purification of tryptic IgG Fc N-glycopeptides. Cotton wool HILIC SPE microtips allowed the removal of salts, most nonglycosylated peptides, and detergents such as SDS from glycoconjugate samples. MALDI-TOF-MS glycopeptide profiles were very repeatable with different tips as well as reused tips, and very similar profiles were obtained with different brands of cotton wool pads. In addition, we used cotton HILIC microtips to purify N-glycans after N-glycosidase F treatment of IgG and transferrin followed by MALDI-TOF-MS detection. In conclusion, we establish cotton wool microtips for glycan and glycopeptide purification with subsequent mass spectrometric detection.     

Study of Eye Drops Dispensing and Dose Variability by Using Plastic Dropper Tips

ABSTRACT

The application of eye drops from flexible dropper bottles fitted with different types of dropper tips is associated with the high variability of eye drop weights. The aim of this report was to investigate the simultaneous effect of three factors influencing the mean weight of drops dispensing from two plastic dropper tips. Using a designed experiment (Box-Behnken), the effect of the concentration of benzalkonium chloride solutions (BAC) in the range of 0–0.02%, the dispensing angle from 90° to 30° from horizontal, and the residual volume of liquid in the dropper bottle from 4 to 10 mL on the mean drop weights were examined. The significant effect of the increase in BAC concentration resulted in a linear decrease in drop weights for both of the dropper tips investigated. The significant effect of the dispensing angle was influenced by the dropper tip design. For the dropper tip A, the effect of the dropper tip tilt was described by the quadratic equation with a minimum, which corresponded to the dispensing angle equal to that of 48° from horizontal. Below this angle, the increase in drop weights occurred due to the drop formation from the wetted external surface of the tip orifice. The linear decrease in drop weights in response to the decrease in dispensing angle was detected for the dropper tip B. The regression equations and the contour line plots obtained allowed the drop weights to be estimated for the actual combinations of both the BAC concentration and the dispensing angle. The effect of the residual volume was found to be non-significant. Based on the formula of Tate's law, the direct proportion between surface tension of a solution and the radius of the effective perimeter of a dropper tip can be used to estimate the theoretical maximal weight of drops at the dispensing angle of 90°. Using the stalagmometric values of surface tension of the BAC solutions, the maximal drop weights were estimated for both of the dropper tips investigated. A comparison between the theoretical and the experimentally measured drop weights enabled the dropper tips behavior to be discussed by using Harkins and Brown correction factor F. The F-value of 0.74 noted for the dropper tip A differed from that of stalagmometer F-value (0.61) indicating a deviation from the simple drop formation process in answer to more complicated design of the dropper tip A. On the other hand, the F-value of 0.6 observed for the dropper tip B demonstrated the better consistency with stalagmometry. As a result, the dropper tip B with the linear decrease of drop weights in response to the increased concentration of BAC and the decreased dispensing angle without the adverse external drop formation could be recommended in real drop dispensing.