In order to make high-performance liquid chromatography (HPLC) more widely available at home and in small-scale settings, we have simplified two of its most costly modules, namely the pump and the detector. This should make the setup affordable for home or small laboratory use.
Journal of Chromatography A 1639 (2021) 461925 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Towards using high-performance liquid chromatography at home Jan Lankelma a,c,∗, Dirck J van Iperen b, Paul J van der Sluis c a Department of Molecular Cell Physiology, VU University Amsterdam, O|2 Lab Building, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands Department of Fine mechanics and Engineering VU - Bèta, VU University Amsterdam, The Netherlands c Foundation for Chromatography at home, Demonstrator Lab, Amsterdam, The Netherlands b a r t i c l e i n f o Article history: Received September 2020 Revised 13 January 2021 Accepted 16 January 2021 Available online 20 January 2021 Keywords: Chromatography at home low-cost HPLC low-cost electrochemical HPLC detector, low-cost HPLC pump quantified self a b s t r a c t In order to make high-performance liquid chromatography (HPLC) more widely available at home and in small-scale settings, we have simplified two of its most costly modules, namely the pump and the detector This should make the setup affordable for home or small laboratory use A manual HPLC pump was constructed so as to fit into a caulk gun from a local hardware store enabling the generation of 100150 bar of pressure In order to limit the pressure drop during the running of a chromatogram, a pulse dampener was developed We further modified the electrochemical detection (ECD) system so as to use a cheap boron-doped diamond electrode with an overlay of thin filter paper, causing an eluent flow over the electrode by wicking and gravity Both the pump and the detector are at least ten times cheaper than conventional HPLC modules Using a home-packed Jupiter R Proteo reversed phase capillary column we show how this low-cost HPLC system generates well resolving chromatograms after direct injection of fresh urine The ECD did not lose its sensitivity during regular use over more than half a year For homovanillic acid (HVA), which is of medical interest, we measured a linear dynamic range of two orders of magnitude, a detection limit of HVA in the injected sample of μM and a coefficient of variation 60 sec Therefore, neither a nanovolume injection valve (corresponding to < sec on INJECT), nor a splitter system [20] should significantly reduce peak broadening in 3.3 Applications The aim of this work is to present a new tool for HPLC analysis at home or in small laboratories Attractive should be the analysis of urine without or with a simple sample pretreatment (e.g solid phase extraction) After direct injection of fresh urine of a healthy J Lankelma, D.J van Iperen and P.J van der Sluis Journal of Chromatography A 1639 (2021) 461925 Fig Analysis of two urine samples, one (A) just before, and one (B) collected at 1.8 h after taking 10 Kalamata table olives on an empty stomach, indicating the conversion of hydroxytyrosol from the olives to homovanillic acid (at arrow) Column length 15 cm 68-year old volunteer the present method generated 5-20 peaks (Fig 9A) As compared to frozen urine, fresh urine samples offer the advantage that no precipitates have to be removed before injection and that there is no oxidation due to storage We focused on the effect of table olives as a key component of a Mediterranean diet HVA was determined after intake of table olives and quercetin tablets After taking 10 pitted Kalamata table olives on an empty stomach, a rapid rise of the peak at the retention time of HVA was detected in the first urine collected around 1.8 h after ingesting the olives (Figs 9A and 9B) This is in accordance with rapid absorption and a metabolic conversion of HT from the olives [33] Subsequent urine samples showed a decline of the HVA peak At retention times in excess of 40 0 sec the chromatogram of the urine of Fig 9B showed no significant peaks (Fig 10), so under the presented chromatographic conditions the system showed to be ready after about h for the next sample Cleaning of the column by the propanol/nitric acid mixture (see above for details) was done about one time per week and columns could be used for at least half a year enabling HVA measurements, comparable to that shown in Figs 9A and 9B The HVA concentration in the first morning urine sample calculated with the standard plot (Fig S2) and adjusted for the injection volume, was in the order of 10 μM Stroe et al showed that the urinary HVA concentration is more than two orders of magnitude higher than the blood concentration [34], presumably by active tubular organic anion transport in the kidney [35] The urine results thereby yield (indirect) information of variations in the low blood concentrations of HVA The content of HT in table olives was measured under the present chromatographic conditions after the following extraction procedure The fruit flesh of three olives was cut into pieces of 23 mm and immersed in 200 ml of tap water overnight The following morning, after stirring a couple of times, the mixture was filtered through cotton wool and the filtrate was directly injected into our chromatographic system (Fig S7) A second extraction of the residue (under the same conditions) yielded less than 5% of the first extraction, indicating that almost all the HT had diffused overnight into the water An additional experiment was the determination of HVA after intake of quercetin After taking an oral dose of 500 mg of J Lankelma, D.J van Iperen and P.J van der Sluis Journal of Chromatography A 1639 (2021) 461925 Fig 10 The chromatogram of Fig 9B at a longer time scale, showing the absence of significant peaks in the later phase 400 quercetin and subsequent collection of urine samples from two healthy volunteers, a peak at the retention time of HVA was emerged much later (around 12 h, data not shown) than after the consumption of Kalamata olives This delay may reflect the intestinal transit time and conversion of quercetin to HVA by the microflora in the large intestine [36] Therefore, the HVA pattern may carry quantitative information on the composition of the microbiome The tool we present here does not identify the peaks at the retention times of HVA or HT as HVA or HT with 100% certainty, even after spiking the sample with pure HVA or HT For our test compound HVA we have compared urinary concentrations with those measured by HPLC-MS-MS The agreement between both methods (Fig 11) confirms the identity of the measured peak to be that of HVA Increasing the length of a home-packed column beyond 15 cm enhanced separation power, with inherently longer elution times (Figs S5 and S6) y = 0.9896x R² = 0.9925 ECD: HVA (µM) 300 200 100 3.4 Perpectives 100 200 300 400 MS-MS: HVA (µM) In an attempt at simplifying HPLC we have integrated a lowcost hand pump, a pulse dampener and a battery-powered detector The estimated total cost of the presented setup amounts to EUR 2200 for off-the-shelf components, including EUR 10 0 for a commercial HPLC injection valve, and excluding the cost of construction labor For other published comparable miniaturized and low-weight HPLC systems [11] at least one HPLC sample valve was needed for sample introduction A refurbished valve could reduce the price of the total setup significantly By comparison, relatively simple isocratic HPLC pumps by which the same chromatogram can be created, range from EUR 50 0 to EUR 10 0 Although our newly constructed modules are relatively simple, instrument construction skills are required The amplifier could simply be constructed by any moderately experienced electronics craftsman, or by using a breadboard for connecting the electronic resistances, op-amps, etc The PEEK pump module can be made using a lathe, or ordered like we did Interesting possibilities may exist in the near future for 3-D printing of the PEEK modules or of the sampling valve Increased demand should lower the price and stimulate commercial activities Increasing quantities of machining of the components can reduce the price drastically In the setup presented here the HPLC valve is the most expensive module If higher Fig 11 Comparison of urinary homovanillic acid concentrations between our HPLCECD method and an established HPLC-MS-MS method A linear trend line was obtained using Excel R For two samples with HVA concentrations of 352.7 μM and 279.5 μM (measured with MS-MS) we measured average HVA concentrations of 364.3 μM and 259.0 μM, respectively; the standard deviations were 8.7 μM (n=3) and 16.3 μM (n=5), respectively Samples of two human volunteers were collected after taking Kalamata olives, mucuna pruriens and quercetin and without taking anything (after overnight fasting) separation power is needed, peak broadening caused by the injection should be reduced Then, a valve with a small stator bore may be needed This will be less available than e.g a second-hand wide bore valve For detecting compounds that are not electrochemically active, a florescence detector or a recently published prototype of a UV LED based detection cell [8] could serve The use of alternative commercially available detectors for capillary LC, also of a contactless conductivity detector, would add EUR 20 0 or more to the cost As HPLC is modular, our pump module or electrochemical detector may also be used to replace a pump or detector in another portable system In case a gradient elution will be needed one may J Lankelma, D.J van Iperen and P.J van der Sluis Journal of Chromatography A 1639 (2021) 461925 chose a different miniaturized system [11] with the electronics for gradient pump control Alternatively, one may chose the setup by Lynch switching to eluents with different compositions during a run [14] A simple variant could be a step-gradient after filling the 20 μL loop with a different eluent at some time point after the loop has been used for timed sample injection Here we have focused on technical innovations In order to promote further developments we advocate an open source model, which may then also be applied to different biomolecules We have here focused only on HVA [37,38], which can be formed from dopamine in the brain and other organs [35,39] HVA also increased after the consumption of an extract of seeds of mucuna pruriens, containing the dopamine precursor L-dopa, which can be used in the treatment of Parkinson’s disease [40] A daily dose of mg of HT and derivatives has been recommended by EFSA [41] for reduction of oxidation of LDL cholesterol [42] A large variation in the reduction of oxidation of LDL cholesterol by HT and derivates in humans has been reported [43-46] Also the content of HT of table olives may vary widely [47,48] Comparison with the peak height of a HT standard we found for some Kalamata table olives that just one olive would already be enough to obtain mg of HT, while we found in Thassos table olives less than 0.2 mg HT per olive, which is in line with earlier findings [47] This work may contribute to facilitating more measurements in this applied setting thereby providing bigger data and thereby more understanding of variables involved in the conversion of HT to HVA, e.g the way of administration and the time of day olives are consumed Besides nutrition as a source for HVA, urinary HVA may be produced by neuroblastomas and phaeochromocytomas [49-51] A further practical application of urinary HVA may be its use as a biomarker that can be measured frequently at home, e.g during tumor treatment We propose measurements after food interventions early in the morning after overnight fasting as a good timing with relatively low influence of other food sources For home procedures packing of fused silica capillaries are preferred over making monolithic columns (simpler and working with less toxic materials) Moreover, most of the polymerbased monolithic columns seem to be unable to efficiently separate small molecules [52] Chromatography of hydrophilic oxidizable urinary components using reversed phase column material has been shown here without organic modifier added to the eluent When analyzing more lipophilic compounds with the present system the use of organic modifiers in the eluent may negatively affect the wicking speed of the eluent alongside the working electrode When the transport becomes too slow, a make-up flow can be added from the upper reservoir, analogous to the principle published before [17] Alternatively the construction of the electrochemical cell may be adapted to make the flow over the electrode independent of the wicking [53] for electrochemical detection, when compared to piston or syringe pumps [11] Due to the “open air” construction, the presented detector is not bothered by gas bubbles that can develop, in case UV detection will be used [11] A radar chart assessing the system according to “BETTER criteria 2020” [11] has been presented (Fig S8) In our present setup, the modules have not yet been placed in a single casing for use in the field Compared to other portable and miniaturized HPLC systems we have reduced the construction skills required to those that will often be available locally Our new Foundation for the promotion of the use of chromatography at home will support local constructions by providing additional information through the Internet The simplicity of our system also comes with limitations For example, when an application requires a gradient elution, other reported systems should be considered The isocratic mode of elution and the direct injection of urine contributed to a long elution time before the next sample could be injected However, for a limited number of samples to be analyzed at home this should not constitute a problem Other detectors could open a new window for detecting compounds, but their acquisition could considerably increase the price of the setup As an application we analyzed fresh urine and focused on one peak that was easily detectable and that showed a large variation during the day This peak was at the retention time of HVA and its concentration in urine was dependent on the intake of HT However, other compounds in the urine may be measured as well Other detectors and superficially porous silica particles or monolithic columns may be used to broaden the scope and speed of analysis of easily measurable components without sample pretreatment Hopefully, this low-cost approach will stimulate more research at home into the physiology of oneself or of dear ones and in the near future it may contribute to managing health Declaration of Competing Interest The authors declare that JL and PvdS are board members of a non-profit Foundation for the promotion of the use of chromatography at home CRediT authorship contribution statement Jan Lankelma: Investigation, Conceptualization, Methodology, Writing - original draft Dirck J van Iperen: Investigation, Resources Paul J van der Sluis: Investigation, Writing - review & editing Acknowledgements We are grateful to Jaap Tijmes and Arthur van de Tetrix for their help with the construction of the PEEK modules Errol Dekkinga of D-Tech Staalbouw has helped with a prototype of the pump module in stainless steel Tom Tijmes is thanked for IT support and colleagues of the VU instrumental development group are gratefully acknowledged for skillful technical advice Martijn van Faassen (Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands) is gratefully acknowledged for analysis by HPLC-MS-MS Henk Dekker is acknowledged for advice on data management We thank colleagues in the nutrition research field for valuable discussions and colleagues in the lab for experimental support Hans Westerhoff is gratefully acknowledged for improvements in the style and structure of the manuscript Conclusions The work presented shows the feasibility of constructing one’s own low-cost HPLC system for urine analysis after food interventions at home Key components are a hand pump, requiring no batteries and capillary LC, in combination with a simple electrochemical detection system with a flow generated by wicking and gravity along a BDD electrode The present system has a low weight (< kg, excl laptop) and is potentially a portable system for measurements in the field, but its robustness will have to be improved for that purpose, especially regarding the electrochemical detector Figures of merit also comprise its relatively easy construction, when compared to other low-weight systems [11], its low price, and its sensitive detection In addition, a stable eluent flow resulting from the presented pump can be regarded as favorable Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.chroma.2021.461925 J Lankelma, D.J van Iperen and P.J van der Sluis Journal of Chromatography A 1639 (2021) 461925 References [23] N.M Devitt, R.E Moran, J.M Godinho, B.M Wagner, M.R Schure, Measuring porosities 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